Muscle biopsy abnormalities are a consistent feature of the collagen vascular disorders, especially polymyositis and der- matomyositis. Established criteria used ...
Polymyositis- Dermatomyositis Diagnostic and Prognostic Significance of Muscle Alkaline Phosphatase D. Cros, MD, C. Pearson, MD, and M. A. Verity, MD
The distribution and intensitv of alkaline phosphatase deposition in 54 patients with dermatomyositis-polymyositis (PM-DM) was analyzed by the enzyme histochemical method. Increased enzyme reactivity of endomysial capillaries was found in 28% of patients, equally distributed between adult onset PM (Group I) and PM-DM with overlap in other connective tissue diseases (Group V). Patients with high endomysial capillary reactivity (R,: 60) responded poorly to steroids, had an increased incidence of rheumatoid factor, and had less fiber degeneration/necmsis in their biopsies. Twenty-two percent of patients demonstrated prominent perimysial phosphatase reactivity localized in newly formed Collagen and fibroblasts. Thirty patients (55%) demonstrated significant numbers of alkalinephosphatase-positive fibers positively correlated with increased fiber degeneration/necrosis, endomysial fibrosis, increased numbers of triglyceride-containing muscle fibers, and NADH tetrazolium reductase hyperreactivity. Minimal overlap between the three enzyme distribution patterns was found. Endomysial capillary activity probably represents endothelial alkaline phosphatase induction analogous to the pattern seen normally in lower mammals (rat, rabbit, guinea pig). Alkaline phosphatase fiber reactivity probably represents a particular phase in fiber regeneration/maturation especially after denervation and is positively correlated with an increased incidence of spontaneous fibrillation potentials in PM-DM. (Am J Pathol 1980, 101:159-176)
THE USE OF ENZYME HISTOCHEMISTRY in the study of muscle biopsy tissue from patients with neuromuscular disorders is well established. Such techniques have allowed for the recognition of new disease entities and strengthened the diagnostic basis of established myopathic and neuropathic disorders. Muscle biopsy abnormalities are a consistent feature of the collagen vascular disorders, especially polymyositis and dermatomyositis. Established criteria used in the diagnosis of polymyositisdermatomyositis (PM-DM) are the finding on biopsy of fiber degeneration, regeneration, necrosis, and a mononuclear infiltrate.`3 We have used the Gomori alkaline phosphatase histochemical reaction routinely in the evaluation of muscle biopsies from patients with susFrom the Department of Pathology (Neuropathology) and Medicine (Rheumatology), Jerry Lewis Neuromuscular Institute, and the Brain Research Institute, UCLA Center for the Health Sciences, Los Angeles, California. D. Cros is presently Chef de Clinique Rhumatologique et des Maladies Neuromusculaires, Hopital de la Timone, Marseille, France. Supported in part by grants from the Muscular Dystrophy Association of America and USPHS Grant HD-05615. Accepted for publication May 6, 1980. Address reprint requests to M. A. Verity, MD, Department of Pathology, UCLA Center for the Health Sciences, Los Angeles, CA 90024. 0002-9440/80/1008-0159$01.00 159 C) American Association of Pathologists
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pected PM-DM. Some unusual patterns of fiber and extrafiber reactivity have been found in a number of these patients. Normal adult human muscle is characterized by a complete absence of alkaline phosphatase reactivity except for presence in some arterioles4 in contrast to the positively stained capillaries and arterioles found in chickens, rabbits, rats, and mice.57 Our aim in this retrospective study was to evaluate the prognostic and diagnostic significance of the histochemical alkaline phosphatase reaction in muscle biopsies from patients with PM-DM. Specifically, we correlated the alkaline phosphatase reactivity found in muscle fibers, endomysial capillaries, or perimysial connective tissue with other pathologic findings in the biopsy and selected clinical and laboratory studies. Materials and Methods Selection of Patients
The clinical records were reviewed of 74 patients with polymyositis or dermatomyositis, including detailed histochemical muscle biopsy studies. Most of the patients were seen at the UCLA Medical Center between 1972 and 1979, and the clinical data leading to the diagnosis of PM-DM were collected and analyzed according to criteria delineated by the UCLA Rheumatology Group.89 Criteria for inclusion in the series were: 1) symmetrical proximal muscle weakness with or without dysphagia; 2) elevation of serum creatine phosphokinase (CPK); 3) the typical rash of dermatomyositis; 4) electromyographic findings of small amplitude, short duration polyphasic motor unit potentials, fibrillation, increased insertional irritability, and spontaneous high frequency discharges; and 5) muscle biopsy abnormalities compatible with the diagnosis usually revealing fiber degeneration, regeneration, necrosis, and a mononuclear infiltrate. Cases in which clinical information was deficient or which did not fulfill the criteria of "definite" or "probable" PM-DM were excluded. Also, cases with an ambiguous history of central or peripheral nervous system involvement (2 cases), endocrinopathy (1 case), and a suggestive family history of muscular dystrophy (2 cases) were excluded. Three cases revealing a predominant eosinophilic inflammatory infiltrate were also eliminated without regard to the clinical picture. Biopses of 54 cases remained for detailed histochemical analysis and clinicopathologic correlation. The 54 cases were put into the subgroups of a recently proposed scheme 9: I Polymyositis (PM) II Dermatomyositis (DM) III Polymyositis or dermatomyositis associated with malignancy (malig PM-DM) IV Childhood polymyositis or dermatomyositis (child PM-DM) V Polymyositis-dermatomyositis with associated connective tissue disorder (overlap group) Patients included in Group V met independent criteria for both PM-DM and other connective tissue diseases. The ARA criteria for rheumatoid arthritis 'o and the criteria proposed by Cohen et al (1971) for systemic lupus erythematosus were used." Systemic sclerosis was diagnosed on the basis of typical cutaneous changes, Raynaud's phenomenon, and other classic features.'2 Pathologic Studies Methods For each biopsy a sample of muscle was fixed in 10% buffered formalin or Bouin's fluid and embedded in paraffin. Longitudinal and transverse sections were cut and stained with
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hematoxylin-eosin or azure B. Other muscle specimens were quenched in isopentane cooled to -180 C in liquid nitrogen. Transverse cryostat sections were subsequently cut and the following techniques used with frozen serial sections': modified Gomori trichrome, PAS, oil red 0, NADH tetrazolium reductase, myofibrillar ATPase, pH 9.4 and after preincubation at acid pH 4.6 and 4.3*313 Alkaline phosphatase activity was demonstrated by a modification of the Gomori method 14 and the simultaneous coupling method using a-naphthyl phosphate and fast blue RR3 Alkaline-phosphatase-positive endomysial capillary density was determined by direct examination of cryostat sections with the use of an ocular grid at 10 x objective magnification. In controls and patients, fields were selected in which the capillary density was highest and the grid covered intrafascicular areas, avoiding perimysium. At least 700 fibers were counted in 3-4 fields. The result (R, ratio) was expressed as the number of alkalinephosphatase-positive capillaries/1000 fibers.'5 Semiquantitative Study of Pathologic Findings
The perimysial alkaline phosphatase reactivity was noted as + or - in biopsies without regard to the magnitude of this change. Similar + or - gradings were applied in the presence of NADH tetrazolium-reductase-hyperreactive fibers, increased intrafiber lipid (oil red 0), and perifascicular atrophy. The alkaline phosphatase reactivity of muscle fibers was graded on a scale of 1 to 3: 1 = 3 and 15 positive fibers in the biopsy. A similar scale was used to grade the degenerative and necrotic fiber changes revealed in the modified trichrome and periodic acid-Schiff (PAS) reactions and regenerative activity observed in the azure A. The degree of inflammation was studied in both frozen and paraffin sections with the use of a 3-step scale: 1 = single focus of inflammatory cells, minor endomysial interstitial inflammation or occasional perifiber inflammation; 5 = multifocal inflammation with inflammatory foci containing more than 25 cells; and 3 = inflammatory changes intermediate between 1 and 5. Nodular inflammation was graded separately and characterized by one or more confluent dense aggregates of lymphocytes containing >100 cells. Statistical Analyses
Nonparametric statistical methods were used to analyze the data. The Welch test and Student t test for unpaired variants were used in most of the analyses. Values are given as the mean ± SD (number of observations).
Results General Features
The 54 patients in this study were divided into 5 groups as defined by the UCLA Rheumatism Group criteria.8 Table 1 indicates the number of patients in each group, sex distribution, age at the time of biopsy, and the duration of the disease prior to biopsy. Female patients predominated in all groups. Patient age at the time of muscle biopsy was similar in all groups except for Group IV, representing the childhood form of dermatomyositis-polymyositis. No significant difference in the duration of disease prior to biopsy was encountered between Groups I (polymyositis) and V (polymyositis-dermatomyositis overlap), but adult-onset dermatomyositis (Group II) revealed a significantly shorter disease duration prior to biopsy (P < 0.02) probably occasioned by the presence of the skin rash early in the evolution of the disease.
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Table 1-Classification of Patients Into UCLA Defined Subgroups
Group I Group II Group lIl Group IV Group V
Male/Female
Age (mean + SD)
Duration of disease prior to biopsy (months) (mean + SD)
5/16 3/4 0/2 2/3 2/17
52.3 ± 15.7 54.8 ± 14.3 49 ± 56 8.8 ± 4.3 45.4 ± 17
36 ± 41 13 ± 15 15 ± 1 10 ± 9 48 ± 59
Groups: I: Polymyositis; II: Dermatomyositis; III: Poly-dermatomyositis with malignancy; IV: Childhood poly-dermatomyositis; V: poly-dermatomyositis with overlap into other connective tissue disease.
Alkaline-Phosphatase-Positive Capillaries in Inflammatory Myopathy
Large vessels and endomysial capillaries were usually unstained by alkaline phosphatase in normal human muscle (Figure 1). Small arterioles were usually reactive. The R1 ratio in control biopsies was 29.4 + 17 (n = 39). Approximately one third of the patients with polymyositis-dermatomyositis revealed an increase in endomysial capillary alkaline phosphatase reactivity (Figure 2). With the use of this parameter of endomysial capillary hyperreactivity it was possible to identify a subpopulation of patients with inflammatory myopathy with significantly increased reactivity, R1 2 60 (Text-figure 1). Did this hyperreactive subgroup represent a unique patient population? We examined the distribution of patients with high R1 in the previously defined groups of the UCLA Rheumatology Service. When analyzed in this way (Text-figure 2) equal numbers of hyperreactive patients 220-
180*
o
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140-
100-
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%
:
0 00
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it
Control
itF_ Inf lammatory
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TEXT-FIGURE 1-Plot of RI ratios in 39 control biopsies and 54 biopsies from patients with polymyositis-dermatomyositis. Solid circles represent patients with significantly abnormal endomysial capillary alkaline phosphatase reactivity (RI 2 60). RI ratio estimated as the number of alkalinephosphatase-positive capillaries/ 1000 fibers.
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TEXT-FIGURE 2-Distribution of 15 patients with a high (260) R1 ratio between the 5 clinically defined subgroups of polymyositis-dermatomyositis. See the text for the definition of
subgroups.
Group
I
I
IlI
IV
V
found in Groups I, II, and V. No patients with high R1 ratios were found in group III (polymyositis-dermatomyositis with malignancy) or Group IV (childhood polymyositis-dermatomyositis). An apparent increased incidence of endomysial alkaline phosphatase reactivity was found in patients with adult-onset dermatomyositis (5 of 8 patients) in marked contrast to the absence of vascular reactivity in childhood dermatomyowere
sitis.
Clinicopathologic Correlations of Patients With Endomysial Capillary Alkaline Phosphatase Hyperreactivity
The mean age at biopsy and duration of disease prior to biopsy was contrasted between patients with normal R1 ratios and those with R1 ratios 60 (Table 2). The mean age of patients with high R1 ratios was significantly greater (P < 0.005) than that of those patients with normal R1 ratios. The lack of endomysial vascular alkaline phosphatase positivity in patients with childhood PM-DM described above might be expected to bias the mean age data for patients with normal and high R1 ratios. RecalTable 2-Endomysial Alkaline-Phosphatase-Positive Capillaries: Correlation With Patient Sex, Age, and Duration of Disease
Normal R1 39 8/31 41.7 ± 20.3t
High R1 (:60) 15 4/11 56.0 ± 12.9
P
Number of patients Sex ratio: M/F NS Age (mean ± SD) P < 0.005 Duration (months) prior to biopsy 35 ±44 43 ± 63 NS * Data entries are presented as mean + SD; otherwise presented as ratios or percentages. Significance between the groups is calculated from Welch nonparametric analysis of chisquare (x2) analysis of percentages. t If Group IV (child PM-DM) is excluded from the normal R1 group, the mean age increases to 46.4 ± 16.0, which differs from the high R1 group at P < 0.02. NS = not significant.
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culation of the data excluding Group IV patients still revealed a significant difference (P < 0.02) between the mean age at biopsy in the normal and high R1 ratio patients. The increase in disease duration prior to biopsy noted in the high R1 group was not statistically significant. We found no correlation between biopsies showing R1 - 60 and the magnitude of inflammation, regeneration, fibrosis, or fiber atrophy (Table 3), but an inverse relation was found between patients with high R1 ratios and the degree of fiber degeneration/necrosis. Fewer patients with a high R1 ratio revealed perifascicular atrophy, presumably because of the inclusion in this group of childhood dermatomyositis previously shown to have no significant increase in R1 ratio. If biopsies with increased R1 ratios represented a subpopulation of patients with PM-DM, then it was of interest to define such a subpopulation with respect to clinical severity, magnitude of weakness, response to steroids, and other laboratory parameters, including CPK, erythrocyte sedimentation rate (ESR), antinuclear antibody (ANA), and rheumatoid factor (Table 4). Such correlations failed to define a specific clinical syndrome and certainly did not suggest a differing disease pattern of weakness or severity. Notable, however, was the reduced response to steroid therapy in those patients with a high R1 ratio (P < 0.01) and an increased incidence of positive rheumatoid factor. Perimysial Alkaline Phosphatase Reactivity in PM-DM
Twelve patients in this series (22%) demonstrated prominent perimysial alkaline phosphatase reactivity. The reaction product was localized in elongated cytoplasmic strands, often but not always accompanied by mononuclear and other inflammatory cells (Figure 3). In all cases there was widening of the perimysial space, and fibroblasts were always identified (Figure 4). There was only minor overlap between patients showing Table 3-Endomysial Alkaline Phosphatase Positive Capillaries: Pathologic Correlations High R1 Normal R1 P (: 60) (< 60) 15 39 Number of patients 3.1 + 1.7 NS 2.8 + 1.5 Inflammation 1.7 + 0.5 P< 0.05 2.3 + 0.8 Degeneration/necrosis NS 0.75 + 0.8 0.73 ± 1.0 Fibrosis Perifascicular 0.1 < P3) of alkaline-phosphatase-positive fibers between the clinical subgroups of polymyositis-dermatomyositis as proposed by the UCLA Rheumatology Group.
.
-
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-
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167
positive f i bers I...
4 -15 positive fibers < 3 positive fibers
No of Cases
10
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Histochemical Studies
on
Alkaline-Phosphatase-Positive Fibers
Fibers undergoing active necrosis or phagocytosis were not alkalinephosphatase-reactive. In many fibers the reaction product was only present in the subsarcolemmal zone (Figures 5 and 7). In such fibers nuclear hypertrophy and internalization was common. In serial section, alkaline-phosphatase-positive fibers often presented a dislocated, "motheaten" reaction with NADH tetrazolium reductase (Figure 8). Although an increased incidence of alkaline-phosphatase-reactive fibers correlated with an increased number of NADH-tetrazolium-hyperreactive fibers, there was no individual fiber correlation between NADH hyperreactivity and alkaline phosphatase positivity (Figures 5-8). An increased occurrence of spontaneous activity on EMG was posiTable 6-Alkaline-Phosphatase-Positive Fibers: Pathologic and Laboratory Correlations in Three Patient Groups
Low (n = 23)
Intermediate (n = 18)
(< 3)
(3-15)
High (n = 13) (> 15)
P*
Inflammation 3.09 + 1.6 3.44 ± 1.4 2.54 ± 1.6 NS Degeneration/necrosis 1.91 ± 0.7 2.11 ± 0.6 2.38 ± 0.6 P< 0.02 Regeneration 1.70 ± 0.6 2.00 ± 0.6 2.08 ± 0.8 NS Fibrosis 0.61 ± 0.7 0.89 ± 0.7 1.38 ± 1.0 P< 0.025 Atrophy 1.35 ± 0.8 1.72 ± 0.5 2.38 ± 0.5 P< 0.01 NADH hyperreactivity (%) 39% 61% 62% P< 0.05 Oil red O(%) 26% 44% 62% P< 0.05 Maximum CPK (x times normal) 8 ± 11 24 ± 49 72 ± 73 P< 0.001 Rheumatoid factor (% positive) 53% 50% 17% P< 0.05 * The statistical difference was computed between low and high patient groups. Data given are derived from semiquantitative analysis as described in Materials and Methods. NS = not significant.
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TEXT-FIGURE 5-Summary diagram to indicate incidence of alkaline-phosphatase-positive fibers, endomysial capillaries, and perimysium in polymyositis-dermatomyositis. The degree of overlap is indicated by stippled areas.
tively correlated with increased numbers of alkaline-phosphatase-positive fibers (P < 0.05). Spontaneous fibrillation potentials in PM-DM are due to segmental fiber necrosis, which isolates a portion of the muscle fiber from its motor endplate segment.'6"7 The incidence of alkaline-phosphatasepositive fibers after experimental denervation 1 suggests that their increased density in PM-DM may be directly related to the intensity of the segmental "denervating" process. Streib et al 19 revealed that the paraspinal muscles were most frequently involved in spontaneous fibrillation potentials. Discussion
Although vascular changes in the inflammatory myopathies have been described previously and include increased thickening and lamination of the basement membrane in arterioles and capillaries, endothelial swelling, and endothelial inclusions,20"24 to our knowledge, changes in histochemical reactivity of the microvasculature have not been analyzed. In this study, approximately a third of the patients with PM-DM demonstrated increased endomysial vascular alkaline phosphatase reactivity. The method of quantitative analysis in which capillary density was expressed/1000 muscle fibers may fail to account for an increased ratio resulting from the loss of fibers through necrosis, atrophy, or fascicular collapse. If this had been the case, we would have found a positive correlation between high RI ratio and increased fiber degeneration, necrosis, fibrosis, or atrophy. We did not, and in fact an inverse correlation was found between high RI ratios and fiber necrosis (P < 0.05). We may infer that the endomysial vascular alkaline phosphatase hyperactivity is an absolute change in the number of reactive vessels. Three possibilities may be envisaged: 1) enzyme induction has occurred in the endothelium; 2) an increase in endomysial capillary density has occurred;
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or 3) there has been endothelial pinocytotic uptake of locally formed alkaline phosphatase. The possibility that interstitially released alkaline phosphatase may be removed pinocytotically by muscle capillaries was demonstrated by Johansson25 in experiments with microinjected horseradish peroxidase. This suggestion seems unlikely, since the increased R1 ratio was not associated with an increase in the number of alkaline phosphatase positive fibers or perimysial connective tissue reactivity. Fell and Danielli 26 observed that the endothelium of newly formed capillaries in experimental wounds was strongly alkaline-phosphatase-positive. Such observations would support the concept of neovascularization after injury in PM-DM, especially since an increase in capillary growth has been demonstrated in chronically stimulated skeletal muscle.27 However, ultrastructural studies 20,21,23 in PM-DM have failed to find a significant increase in capillary density; and Norton 22 found a decrease in microvascular density in dermatomyositis, SLE, and scleroderma, confirmed by Jerusalem et al.' Finally, if neovascularization occurred, it would be difficult to account for the reduction of blood flow found by Paulson et al 28 in patients with PMDM. The remaining hypothesis, that the endomysial capillary hyperreactivity is due to the induction of alkaline phosphatase enzyme in preexisting capillaries, receives some support from the finding of endothelial hypertrophy, increased endoplasmic reticulum, and mitochondrial mass in capillaries of the inflammatory myopathies.' The stimulation of such selective enzyme induction is unknown, but Jerusalem et al 23 suggested that the ultrastructural findings were consistent with repeated cycles of capillary degeneration and regeneration. Such cyclic change in capillary structure may lead to ontogenic dedifferentiation, and the capillary structure would then closely approach that of lower mammals (rat, mouse, rabbit, guinea pig), characterized by endomysial vascular alkaline phosphatase reactivity.6 While the histochemical findings would be consistent with this hypothesis, they fail to account for the absence of microvascular reactivity in the majority of patients with PM-DM. Of the 15 patients with high RI ratios, 5 were found in Subgroup II (adult dermatomyositis). The high incidence of capillary alkaline phosphatase reactivity in adult-onset dermatomyositis was significantly different (X2 analysis) from the incidence in Group I and may provide a diagnostic indicator in the evaluation of biopsies from patients in either subgroup. The high incidence of capillary reactivity in subgroup II contrasted with the absence of such changes in Subgroup IV (childhood PM-DM) in which 4 of the 5 patients had dermatomyositis. We expected a priori to find abnormalities in endomysial capillary reactivity in the childhood subgroup, because vascular changes are a significant feature of childhood dermato-
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myositis.29,30 However, Carpenter et al 15 demonstrated a decrease in muscle capillary density in childhood dermatomyositis, and such absolute reduction in density may account for the absence of a corresponding increase in R1 ratios in this patient subgroup. The age at biopsy of patients with high R1 ratios was 56 years and significantly greater (P < 0.005) than the patient group with normal R1. In our control series we found no relationship between R1 and patient age, and hence the increased patient age cannot explain the higher R1 ratio. Fiber degeneration and necrosis was significantly less in the high R1 group and correlated with the lower CPK values found in patients with high R1 ratios, although the difference in CPK levels between the two groups was not significant at the P < 0.05 confidence level. The incidence of positive rheumatoid factor serologic tests was higher in patients with an increased R1 ratio. Moreover, patient response to steroid therapy in this group was significantly less than the normal R1 group. The coupling of a high R1 ratio with positive rheumatoid factor serologic tests in patients with PM-DM provides a prognostic measure of the steroid response. The recognition of "steroid-resistant" forms of polymyositis has stimulated the introduction of other forms of chemotherapy, especially immunosuppressive agents.31'32 The recognition of specific patterns in the muscle biopsy correlated with steroid responsiveness will allow for more meaningful therapeutic intervention early in the disease and provide a stronger indication for the introduction of adjunct immunosuppressive regimen. Perimysial alkaline phosphatase reactivity was a characteristic finding in approximately one quarter of patients with PM-DM. There was minimal overlap between biopsies showing perimysial alkaline phosphatase and those showing endomysial capillary reactivity, as only 5% of patients with perimysial change had accompanying increased R1 ratios (also see Text-figure 5). Although perifascicular fiber atrophy and degeneration was a notable finding in dermatomyositis 3'30 only 1 patient in the adult and childhood dermatomyositis subgroups combined demonstrated perimysial reaction. The perimysial reactivity did not represent oblique sectioning of alkaline-phosphatase-positive blood vessels but was more consistent with elongated cellular structures, ie, fibroblasts and attenuated newly formed collagen bundles shown to have increased alkaline phosphatase activity.26,33 34 35 The phosphatase-positive fibroblast proliferation and collagen deposition in PM-DM was an unusual connective tissue reaction; we have not seen alkaline-phosphatase-positive connective tissue in Duchenne muscular dystrophy, the limb-girdle dystrophies, or experimental ischemic myopathy,30 conditions characterized by considerable endomysial and perimysial connective tissue proliferation. Moreover, Robertson et
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al 3 in a study of fibrous capsule formation around the kidney, did not find alkaline phosphatase activity associated with collagen fiber synthesis. Active lymphocytic infiltration and/or nodular lymphocytosis was significantly increased (P < 0.05) in those biopsies characterized by perimysial alkaline phosphatase reaction. The inflammatory infiltrates were not selectively localized in the perimysial compartment and therefore cannot be considered to directly contribute to the enzyme hyperreactivity. Engel and Cunningham 7 found alkaline-phosphatase-positive muscle fibers in a variety of neuromuscular disorders and speculated that the intrafiber enzyme might indicate early degenerative or regenerative change. Benitez-Bribiesca and de la Vega 18 confirmed these findings but also revealed their occurrence at certain stages after denervation. We found a significant number (>3) of positive fibers in 30 (55%) of our cases of PMDM. Increased fiber alkaline phosphatase positivity correlated with an increased incidence of degeneration and regenerative change, fibrosis, atrophy, and NADH tetrazolium reductase hyperreactivity. If the fiber phosphatase reactivity signified a degenerative change, we would expect increased CPK levels, which were in fact found. However, the increased incidence of alkaline-phosphatase-positive fibers in denervation 7,18 is difficult to reconcile with the degeneration/regeneration hypothesis, and we were unable to demonstrate azure A activity and phosphatase activity in the same fibers. We think a more likely explanation is that the positive fibers represented a particular phase, or narrow "window," in fiber regeneration or maturation, especially after denervation. Malotra et al 38 suggested that a trophic factor(s) regulated the alkaline phosphatase level in normally growing muscle and that denervation, by virtue of a loss of trophic influence, would allow for derepression and biosynthesis of enzyme. Of interest in this regard is the demonstration of myophosphorylase in regenerating muscle fibers from patients with McArdle's disease.39'40 Evidence has been presented that the myophosphorylase in these regenerating fibers represents a fetal isoenzyme and can be found in cultures from normal adult muscle.4' The occurrence of alkaline phosphatase may also represent a fetal isoenzyme induction. This retrospective study has revealed considerable heterogeneity in the histochemical alkaline phosphatase reactivity of muscle biopsies from patients with PM-DM. Enzyme reactivity fell clearly into three patterns, viz, significant fiber staining, increased endomysial capillary reactivity, and deposition in the perimysium. The finding of increased phosphatase activity in muscle samples by quantitative analysis 42 must be viewed with caution in patients with inflammatory myopathies. The heterogeneity and compartmentation of activity precludes a meaningful interpretation of
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the quantitative data. Overlap between these three major patterns of phosphatase reactivity was small, and in no instance was complete overlap noted (Text-figure 5). The UCLA Rheumatology classification into five clinical subgroups did not correlate with specific distinctive features in muscle biopsies. References 1. Walton JN, Adams RD: Polymyositis. Baltimore, Williams & Wilkins, 1958 2. Adams RD, Denny-Brown D, Pearson CM: Diseases of Muscle: A Study in Pathology. New York, Harper & Row, 1962 3. Dubowitz V, Brooke MH: Muscle Biopsy: A Modern Approach. London, W. B. Saunders, 1973 4. Romanul FCA, Bannister RG: Localized areas of high alkaline phosphatase activity in endothelium of arteries. Nature 1962, 195:611-612 5. Romanul FCA: Capillary supply and metabolism of muscle fibers. Arch Neurol 1965, 12:497-509 6. Ashmore CR, Doerr L, Somes RG, Jr: Microcirculation: Loss of an enzyme activity in chickens with hereditary muscular dystrophy. Science 1968, 160:319-320 7. Engel WK, Cunningham GG: Alkaline phosphatase-positive abnormal muscle fibers of humans. J Histochem Cytochem 1970, 18:55-57 8. Bohan A, Peter JB: Polymyositis and dermatomyositis. N Engl J Med 1975, 292:344-347, 403-407 9. Bohan A, Peter JB, Bowman RL, Pearson CM: Computer-assisted analysis of 153 patients with polymyositis and dermatomyositis. Medicine 1977, 56:255-286 10. Ropes MW, Bennet GA, Cobb S, Jacox R, Jessar RA: Revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Disease 1958, 9:175-176 11. Cohen AS, Reynolds WE, Franklin EC, Kulka JP, Ropes MW, Shulman LE, Wallace SL: Preliminary criteria for the classificaiton of systemic lupus erythematosus. Bull Rheum Dis 1971, 21:643-648 12. Rodnan GP: Scleroderma, calcinosis and eosinophilic fascitis, Arthritis and Allied Conditions: A Textbook in Rheumatology. Edited by DJ McCarthy. Philadelphia, Lea & Febinger, 1979, pp 762-809 13. Brooke MH, Kaiser KK: Muscle fiber types: How many and what kind? Arch Neurol 1970, 23:369-379 14. Barka T, Anderson PJ: Histochemistry: Theory, Practice and Bibliography. New York, Hoeber, 1963 15. Carpenter S, Karpati G, Rothman S, Watters G: The childhood type of dermatomyositis. Neurology 1976, 26:952-962 16. Desmedt JD, Borenstein S: Relationship of spontaneous fibrillation potentials to muscle fiber segmentation in human muscular dystrophy. Nature 1975, 258:531-534 17. Henriksson KG, Stalberg E: The terminal innervation pattern in polymyositis: A histochemical and SFEMG study. Muscle Nerve 1978, 1:3-13 18. Benitez-Bribiesca L, De la Vega G: Occurrence of alkaline phosphatase-containing muscle fibers after denervation. J Histochem Cytochem 1971, 19:691-692 19. Streib EW, Wilbeurn AJ, Mitsumoto H: Spontaneous electrical muscle fiber activity in polymyositis and dermatomyositis. Muscle Nerve 1979, 2:14-18 20. Shafiq SA, Milhorat AT, Gorycki MA: An electron-microscope study of muscle degeneration and vascular changes in polymyositis. J Pathol Bacteriol 1967, 94:139-147 21. Gonz'alez-Angulo A, Fraga A, Mintz G: Submicroscopic alterations in capillaries of skeletal muscles in polymyositis. Am J Med 1968, 45:873-879 22. Norton WL: Comparison of the microangiopathy of systemic lupus erythematosus, dermatomyositis, scleroderma and diabetes mellitus. Lab Invest 1970, 22:301-308
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Acknowledgments We gratefully acknowledge the assistance of Margaret Hall and Laurel Reed in the preparation of the histochemical procedures and the numerous physicians and surgeons involved in patient care who submitted detailed follow-up information.
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Figure l-Quadriceps muscle biopsy from control patient (Rl = 21) revealing no endomysial capillary alkaline phosphatase reactivity. (Gomori technique, x 1 10) Figure 2C-Quadriceps biopsy specimen from a patient with adult polymyositis, revealing an increased (260) R1 ratio (endomysial alkaline-phosphatase-positive capillaries). (Gomori technique, x 110) Figure 3-Perimysial pattern of alkaline phosphatase reactivity in polymyositis-dermatomyositis. (Gomori technique, x65) Figure 4-Detail of interfascicular perimysial alkaline phosphatase reactivity in adult dermatomyositis. Note the absence of endomysial vascular or fiber staining. (Gomori technique, x300)
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Figure 5-Gomori alkaline-phosphatase-positive fibers (arrows). (x250) Figure 6-Serial section compared with Figure 5, stained with the NADH2 tetrazolium reductase reaction. Note that the alkaline-phosphatase-positive fibers (arrows) reveal intermediate dehydrogenase reactivity. Rare angular NADH2 tetrazolium-reductase-hyperreactive fibers are noted elsewhere. (x 200) Figure 7-Alkaline-phosphatase-positive fibers in advanced chronic polymyositis revealing angular and rounded forms with variable intrafiber reaction product. (x250) Figure 8-Serial section for comparison with Figure 7. NADH2 tetrazolium reductase demonstrates ".moth-eaten" features in many fibers and variability of reaction in alkaline-phosphatase-positive fibers (arrows). (x250)
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