The Nature of Myospherulosis of the Upper Respiratory Tract JUAN ROSAI, M.D.
KYRIAKOS recently reported 16 cases of a disease involving the paranasal sinuses, nose and middle ear, which he called "myospherulosis." 5 This was quickly followed by an electron-microscopic account of the condition 1 and its experimental production in animals. 2 The authors related the disorder to the use of hemostatic packing containing petrolatum-based ointments and gauze, after they found that all of their patients had had previous surgical procedures in the region, at which time such a material had been introduced. This astute observation was confirmed experimentally by the reproduction of the disease in animals, and the culprit agent was identified as the petrolatum-based antibiotic ointment. Microscopically, the condition was characterized by the presence of tissue spaces containing scattered amorphous debris, as well as peculiar brown sac-like structures ("parent bodies") which were enclosing numerous "endobodies" or "spherules." A granulomatous inflammatory reaction was often seen surrounding these formations. After consultation with Dr. Daniel H. Connor at the Armed Forces Institute of Pathology, Washington, D. C , it was ascertained that the structures in question had microscopic features similar to those seen in a disease previously found in Kenya and Uganda, known as "myospherulosis" or "subcutaneous spherulocystic disease." 4 7 8 The morphologic similarities of the two conditions were confirmed by the St. Louis authors,
Received November 11, 1977; accepted for publication November 29, 1977. Address reprint requests to Dr. Rosai: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Box 609 Mayo Memorial Building, 420 Delaware St., S. E., Minneapolis, Minnesota 55455. 0003-9173-78-0500—0475$00.85 © Ar
475
Division of Surgical Pathology, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota
who therefore adopted the name "myospherulosis" for their own cases. It is clear from their description that the nature of these structures puzzled them a great deal. The possibility that they represented "some type of fungus" seemed to attract them the most, and this was reflected in the descriptive terminology they employed, such as fungal-like and sporangium-like. They compared the morphologic features of their cases with those of rhinosporidiosis and coccidioidomyosis, and established that despite some superficial similarities, there were substantial differences among them. Furthermore, the negative staining reaction of the structures in question with Gomori's methenamine silver (GMS) and periodic acid-Schiff (PAS) and their uniform failure to grow in cultures did not support a fungal etiology. They concluded that the nature of the structures was unsettled, but suggested that they had been introduced by the antibiotic ointments. In that regard, they pointed out that these medications are not required to be sterilized (implying that they might be contaminated by the organisms) and recommended a temporary ban of antibiotic ointments or petrolatum-based gauze as packing material. 5 The notable occurrence of 16 patients with this disease in one medical center in a relatively short time was emphasized, as well as the dismay of the authors upon finding that every example in their series had been overlooked by the surgical pathologist who had initially examined the tissue sections. We recently had the opportunity to study a case of this condition occurring in the maxillary sinus of a child. This has given us the opportunity to evaluate these mysterious formations, to perform a series of special staining reactions on them, and to carry out a very simple experiment, which we believe throws some light on their nature. Report of a Case An 8-year-old boy was seen at a hospital in Omaha, Nebraska, because of a mass in the lower aspect of the left orbit. Roentcan Society of Clinical Pathologists
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Rosai, Juan: The nature of myospherulosis of the upper respiratory tract. Am J Clin Pathol 69: 475-481, 1978. A case of myospherulosis of paranasal sinuses similar in appearance to those recently seen in St. Louis, Missouri, was examined to ascertain the nature of the sac-like structures seen in this disease. The morphologic features of these formations and their positivity with stains for hemoglobin, peroxidase, and lipofuscin strongly suggest that they represent collections of erythrocytes altered by a foreign substance. This interpretation was confirmed by experimental production of these structures by the action of tetracycline ointment on a pure preparation of human erythrocytes. (Key words: Myospherulosis; Erythrocytes.)
ROSAI
476
genograms of the region demonstrated changes consistent with a soft-tissue tumor, with extension into the maxillary bone. An exploration was performed and biopsies of the maxillary sinus and bone were taken. At the conclusion of the procedure, the cavity was packed with gauze impregnated with 3% tetracycline ointment (Achromycin*). Following a pathologic diagnosis of fibromatosis, a radical maxillectomy was performed. The interval between the biopsy and the operation was 13 days. A local recurrence developed in the skin of the cheek overlying the area of the previous excision 5 months after the surgical procedure and was excised. The patient remains free of disease 5 months later.
Material and Methods
Results Sections from the first biopsy and the late skin recurrence showed features of fibromatosis. Sections from the maxillectomy specimen stained with hematoxylineosin showed, in addition to the fibromatosis, numerous tissue spaces of large size. Some were surrounded by a fibrous stroma and others by a granulomatous reaction composed of histiocytes and foreign-body giant cells. A marked lymphoplasmacytic inflammatory response was present beneath the respiratory epithelium. Some of the spaces were empty; others contained a variety of colorless amorphous debris with the appearance of foreign bodies, as well as numerous sac-
like structures identical to those described in the previously mentioned articles, as judged from their description and illustrations (Fig. \A). They ranged in size from 20 to 100 jum. The majority were lying free in the spaces; a few were attached to the wall and gave the impression of actually arising from it. Their shape was round or oval and their color varied from pale eosinophilic to dark brown. Most of the structures were various shades of brown; in some this color was so intense and dark as to almost obscure the inner structure. Finally, some formations (usually of smaller sizes than the preceding ones), were uniformly dark brown, without discernible inner structures; these corresponded to the "brown bodies" of the St. Louis authors. 5 Close examination of the sac-like formations showed that they had in their interiors variable numbers of spherules, measuring 5 /tm on the average (Fig. IB). In contrast to the extreme variations in size of the sacs, these spherules were relatively uniform in size, the range being from 4 to 7 /j,m. Their numbers per sac ranged from 2 or 3 to more than a hundred. Most of these spherules had a round configuration, but others were collapsed and adopted a concave shape; indentations and other irregularities of the membrane were common. Their content was invariably light, structures resembling nuclei being uniformly absent. The spherules staining a pale eosinophilic color had a thin, smooth membrane quite reminiscent of that of erythrocytes; those that had a brown color were noticeably thicker and coarser, with an irregular granularity on the outside. These findings suggested the deposition of a foreign material onto a pre-existing biologic structure. The appearance of the wall of the sac recapitulated that of the inner structures, in terms of both thickness and staining reactions. When cut tangentially, a coarse granularity could be appreciated. Examination of unstained sections mounted in glycerin-water under high contrast conditions (obtained with a dark field or by simply lowering the condenser in a standard bright field) revealed that some of the sac-like structures had a thin, sharply defined membrane, whereas others had a distinct double-layered membrane measuring approximately 1 pm thick (Fig. 1C). The appearance was remarkably similar to that of the erythrocytes present elsewhere in the section. Periodic acid-Schiff, Mayer's mucicarmine, and Giemsa stains were uniformly negative. Gomori's methenamine silver was largely unreactive; however, a few of the structures showed a definite membrane stain of the spherules; in appearance and intensity of the reaction they were similar to occasional clumps of erythrocytes present elsewhere in the specimen. The iron stain was negative for the sacs and their con-
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Microscopic slides from the radical maxillectomy specimen were stained with hematoxylin-eosin, periodic acid-Schiff (PAS), Gomori's methenamine silver (GMS), Mayer's mucicarmine, Giemsa, iron (Perls' stain), lipofuscin (Schmorl's stain), hemoglobin (Okajima's stain) and peroxidase (diaminobenzidine stain). 3 ' 6 ' 10 In an attempt to reproduce the structures seen in the tissues, the following experiment was performed: the inside bottom of a series of test tubes was coated with a thick layer of 3% tetracycline ointment (Achromycin®); 0.5 ml of a fresh preparation of packed human erythrocytes was added to each tube and the preparations were incubated in a water bath at 37 C, with or without shaking, for 1, 2 and 5 days. Following incubation, a portion of the mixture was smeared in a glass slide, fixed in absolute ethanol, and stained with hematoxylin-eosin. Another portion was admixed with thrombin, 9 dehydrated, embedded in paraffiin, sectioned, and stained with the same battery of reagents used for the operative specimen. Control preparations, which were processed and stained in an identical fashion, included (1) 3% tetracycline ointment taken out directly from the dispenser tube; (2) 3% tetracycline ointment after incubation at 37 C for the same periods as above; (3) fresh packed human erythrocytes after incubation at 37 C for the same period as above.
A.J.C.P. . May 1978
NATURE OF MYOSPHERULOSIS
Vol. 69 • No. 5
Microscopic examination of the smears and microscopic slides prepared from the mixture of tetracycline ointment and human erythrocytes showed the presence, among collections of unremarkable erythrocytes and clumps of amorphous colorless material, of structures recapitulating in most respects the appearance of those seen in the maxillary sinus (Fig. 2). This applied to their size, shape, morphology and all of the staining reactions mentioned above. The only major difference was that, in sections stained with hematoxylin-eosin, most of these structures were eosinophilic; only a few showed the brown color seen so prominently in the operative specimen. The wall was thin and smooth in some spherules, and thick and coarsely granular in others. Again, their appearance was strikingly reminiscent of that seen in the human material. This observation particularly applied to the unstained sections examined under high-contrast conditions. These structures were present in all the experiments, regardless of the time and type of the incubation and method of preparation of the specimen, although they were seen in greater number and better preservation in the 24-hour sample. Sections and smears from the erythrocyte controls (without tetracycline ointment) showed only erythrocytes in a good state of preservation, without clumping or brown discoloration. Sections and smears from the tetracycline ointment (without erythrocytes) showed only an amorphous colorless material. Discussion We were just as puzzled as our St. Louis colleagues about the nature of the sac-like structures when we detected them in the case here described. Our first
impression, like theirs, was that they represented some type of fungus. Yet, we had the same inability to relate them to any known type of pathogenic fungus and to explain, if they were indeed fungi, why they would not stain with the PAS reaction or grow in the appropriate cultures. We also considered the possibility of pollen grains or other vegetable matter, but regarded it as unlikely in view of their morphology and alleged negative staining reaction to cellulose. 5 We also looked for help outside our department and showed the slides to microbiologists, parasitologists and experts in microscopic identification of particles. None of them could identify the structures, but we found amusing and eventually intriguing their recurrent comment that the spherules within the sac resembled erythrocytes. We had made that observation ourselves, but not elaborated further on it in view of the brown color of the spherules and the observation that they were contained within a sac. The fact that several other observers were struck by that similarity led us, finally, to ask ourselves this very simple question: is it possible that the reason the spherules resemble erythrocytes so much is simply that they are erythrocytes, altered by the action of the antibiotic ointment? That certainly would have explained their negativity with PAS and other special stains, lack of nuclear structure, and failure to grow in culture media. Even the equivocal stain seen with Gomori's methenamine silver stain could be explained, inasmuch as some of the erythrocytes present elsewhere in the sections reacted similarly. We then proceeded to perform the abovedescribed stains and experiment, the results of which strongly suggest that this is indeed the case. The unequivocal positivity of most of the structures with Okajima's stain gave the first strong support to this hypothesis, since this reaction is regarded as specific for hemoglobin.10 That other structures did not stain with this procedure may be interpreted as the result of leakage of hemoglobin in severely damaged cells. The strong positive reactions obtained with the peroxidase and lipofuscin stains strengthened this impression. However, the most convincing evidence for this interpretation was that we were able to reproduce the structures by the action of the antibiotic ointment on a pure preparation of human erythrocytes. Close examination of the many structures formed in this experiment gave a clue as to their genesis. The affected erythrocytes clumped in groups of various sizes; most of them seemed to lose their biconcave shape in the process, as suggested by the fact that most of them had a round shape in a section in which they were expected to be randomly oriented. A spectrum was found, ranging from spherules with a thin eosinophilic membrane to others with a brown thick membrane containing irregular deposits
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tents, but stained strongly some coarse clumps of amorphous material present in the cytoplasm of multinucleated giant cells. With Okajima's hemoglobin stain, most of the saclike structures showed an unequivocal positive reaction, paler but otherwise identical to that seen in the erythrocytes present in the specimen (Fig. ID); only a few of the formations remained negative. The diaminobenzidine reaction for peroxidase was strongly positive in all the sac-like structures and similar in every respect to that seen in adjacent erythrocytes (Fig. IE). Whenever one of the two above-mentioned reactions was positive in a given structure, this was true both for the spherules and for the sac that contained them. With Schmorl's lipofuscin stain, some of the structures stained pale blue, similar to erythrocytes; other formations (especially the "brown bodies") showed a much stronger, dark blue positive reaction in the interior of the sac between the spherules (Fig. I F ) .
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FIG. 1. Myospherulosis of paranasal sinus. A, several sac-like structures containing numerous spherules can be seen in a large tissue space, one of them attached to the wall. Hematoxylin and eosin. x500. B, higher magnification of two sac-like structures. The lower one is thick-walled and dark brown; the upper one is thick-walled, pale, and eosinophilic. Hematoxylin and eosin. x 1,250. C, unstained section, x 1,250. D, Okajima's hemoglobin stain, x 1,250. E, diaminobenzidine stain, x 1,250. F, Schmorl's stain, x 1,250. The ill-defined clear round areas within the sac correspond to the spherules.
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FIG. 2. Sac-like structures formed by the action of tetracycline ointment on a pure preparation of human erythrocytes. A, hematoxylin andeosin. x500. ,8, hematoxylin andeosin. x 1,250. C, unstained section, x 1,250. D, Okajima's hemoglobin stain, x 1,250. £ , diaminobenzidine stain, x 1,250. F, Schmorl's stain. x750.
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A.J.C.P. • May 1978
present in the sections were empty, and thus resembled the spaces seen in other conditions associated with accumulation of abnormal lipids, such as sclerosing lipogranuloma. Even when the sac-like structures were present, they occupied only a small area in the cavity. This suggests that the spaces were originally filled with the oily material used as the carrier for the antibiotic, which had been removed during the tissue processing. This by itself is a possibility worth investigating further. The final consideration involves the possibility that these conclusions could also apply to the African cases. Since we have not had the opportunity to examine them personally or to perform special stains on them we cannot establish it. There are, however, some features that suggest that this might well be the case. Their morphology is said to be identical to that in the American cases, 5 except for their color, which might be a function of the specific agent inducing the change. Hutt and colleagues, 4 in their microscopic description of the cases from Uganda, repeatedly commented upon size and other similarities between the spherules and erythrocytes. They even suggested that the disease might have been produced by the injection of foreign substances, in view of the fact that most of them appeared at "popular injection sites." It is hoped that individuals having access to this material will examine this proposition by performing the same staining reactions that we have applied to the American disease described by Kyriakos.
We did not address ourselves to the question of which component in the antibiotic ointment is responsible for this phenomenon. We suspect it is the petrolatum base rather than the antibiotic, but will let others (perhaps the manufacturers of the medicament) work out the specifics of this biologically intriguing phenomenon. The evidence provided in this article in the sense that the structures in question are not microorganisms of any kind, but rather badly battered erythrocytes, answers the practical questions raised in previous publications about the reasons for the mysterious appearance of an African disease in the American midwest, the possible contamination of antibiotics with microorganisms (for which we found no evidence), and the suggestion of banning their use (which may not be necessary after all). Actually, that these structures have been overlooked until now does not seem, in retrospect, to have been of grave consequence. The fact still remains that an inflammatory reaction of foreign-body type was often present around the formations. We suspect that this reaction was directed not against the clumped erythrocytes but against the foreign material introduced with the antibiotic. In this regard, it may be significant that many of the large tissue spaces
Acknowledgments. The following individuals, all but one from Minnesota, examined the microscopic slides and/or were otherwise helpful in the performance of this study: Dr. Donna Blazevic. Mr. Ray G. Bowman, Dr. Richard Brunning, Mr. Edward Cushing, Dr. Louis P. Dehner, Mr. Barton P. Epstein. Dr. Richard D. Estensen, Ms. Barbara Evans, Ms. Lilian Huehnert. Mr. James Jagodzinski, Dr. Donald Larson, Dr. Jeffrey McCullough. Dr. Edward B. Price (Omaha, Nebraska). Dr. Walter Runge. Ms. Joanne Samuelson, Dr. Arthur Sanders, Mr. James Tucker. ADDENDUM
Following the submission of this manuscript, additional data were obtained which tend to substantiate the opinions expressed in the text. Paraffin blocks of two cases of myospherulosis from Uganda were obtained through the kindness of Dr. Alexander C. Templeton, The St. Vincent Hospital, Worcester, Mass., Prof. M. S. R. Hutt, Dept. of Morbid Anatomy, St. Thomas Medical School, London, England, and Prof. R. Owor, Dept. of Pathology, Makerere University, Kampala, Uganda. Examination of hematoxylin-eosin sections confirmed the original descriptions as well as the marked similarity with the American cases, except for the color of the formations, which were pale eosinophilic instead of brown. The Okajima's hemoglobin stain and the diaminobenzidine reactions were both strongly positive in many of the formations, thus indicating that the "myospherules" of the African cases are also made up of red blood cells.
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on the outside; this was interpreted as the result of a progressive alteration due to increasing deposition of a foreign material on them. The brown discoloration was attributed to the accumulation of decomposition products of hemoglobin rather than to a direct impregnation by the antibiotic ointment, which was colorless in smears and sections. The positivity with the Schmorl's reaction for lipofuscin would seem to support this interpretation. That there was a greater proportion of brown structures in the specimen than in the experiment could be a function of time, which was 13 days in the former and only 5 days or less in the experiment. It was difficult to decide whether the envelope was made up by erythrocytic membranes or exclusively by some foreign material. It seems difficult to believe that erythrocytic membranes can rearrange themselves to make a continuous sphere 100 urn in diameter or more. Yet, the positive reaction of this envelope with the hemoglobin, lipofuscin and peroxidase stain and its alleged morphologic similarity to the membrane of the spherule by electron microscopy 1 suggest that this is indeed the case. Incidentally, the ultrastructural description provided by De Schryver-Kecskemeti and Kyriakos 1 is consistent with our interpretation. Even if the authors do not clearly state it, it is obvious from their electron micrographs that the walls of the spherules and envelope are not normal biological membranes of any sort and that, if they are membranes at all, their inner structure has been totally obscured by the deposition of an extraneous material.
Vol. 69 • No. 5
NATURE OF MYOSPHERULOSIS
481 References
1. De Schryver-Kecskemeti K, Kyriakos M: Myospherulosis. An electron-microscopic study of a human case. Am J Clin Pathol 67:555-561. 1977 2. De Schryver-Kecskemeti K, Kyriakos M: The induction of human myospherulosis in experimental animals. Am J Pathol 87:33-40, 1977 3. Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal convoluted tubules of the mouse kidney: Ultrastructural cytochemistry by anew technique. J HistochemCytochem 14:291-302, 1966 4. Hutt MSR, Fernandes JJ, Templeton AC: Myospherulosis (subcutaneous spherulocystic disease). Trans R Soc Trop Med Hyg 65:182-188. 1971 5. Kyriakos M: Myospherulosis of the paranasal sinuses, nose and middle ear. A possible iatrogenic disease. Am J Clin Pathol 67:118-130, 1977 6. Luna LG (editor): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third edition, New York, McGraw-Hill, 1968 7. McClatchie S, Bremner AD: Unusual subcutaneous swellings in African patients. East Afr Med J 46:625-633. 1969 8. McClatchie S, Warambo MW, Bremner AD: Myospherulosis. A previously unreported disease? Am J Clin Pathol 51:699704, 1969 9. Naylor B, Smith MJ: Techniques of cytopathology. Ann Arbor, University of Michigan, 1970 10. Okajima K: On the selective staining of the erythrocyte. Anat Rec 11:295-296, 1916
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An additional feature of interest in one of these cases was the presence of sacs similar in size and shape to those with the "spherules" but containing instead degenerated neutrophils. Prof. Hutt commented that he has recently seen a "definite case in a senior doctor working in the States. This was a lesion in the buttock and it was of interest that he had, at one time, injected himself with a rather dubious antibody when he was traveling in a country with poor medical care." Dr. Stephan A. Szumski, of Lederle Laboratories, Pearl River, N.Y., made the following comment in regard to the brown color seen in the American cases, that we had interpreted as probably resulting from the accumulation of decomposition products of hemoglobin: "The decomposition product in all probability is acid hematin, the same product produced by the action of dilute hydrochloric acid on erythrocytes. It was the basis for the old test used to determine hemoglobin levels in the blood. In that test, a measured amount of blood was added to dilute HC1 and the brown color (acid hematin) produced was measured by comparison with a tinted glass standard. We would expect that this property of HC1 would be retained by the tetracycline hydrochloride in the ointment. The latter is a salt of a weak base, tetracycline, and a strong acid, HCI, the pH is approximately 2.0."
KYRIAKOS recently reported 16 cases of a disease involving the paranasal sinuses, nose and middle ear, which he called "myospherulosis." 5 This was quickly followed by an electron-microscopic account of the condition 1 and its experimental production in animals. 2 The authors related the disorder to the use of hemostatic packing containing petrolatum-based ointments and gauze, after they found that all of their patients had had previous surgical procedures in the region, at which time such a material had been introduced. This astute observation was confirmed experimentally by the reproduction of the disease in animals, and the culprit agent was identified as the petrolatum-based antibiotic ointment. Microscopically, the condition was characterized by the presence of tissue spaces containing scattered amorphous debris, as well as peculiar brown sac-like structures ("parent bodies") which were enclosing numerous "endobodies" or "spherules." A granulomatous inflammatory reaction was often seen surrounding these formations. After consultation with Dr. Daniel H. Connor at the Armed Forces Institute of Pathology, Washington, D. C , it was ascertained that the structures in question had microscopic features similar to those seen in a disease previously found in Kenya and Uganda, known as "myospherulosis" or "subcutaneous spherulocystic disease." 4 7 8 The morphologic similarities of the two conditions were confirmed by the St. Louis authors,
Received November 11, 1977; accepted for publication November 29, 1977. Address reprint requests to Dr. Rosai: Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Box 609 Mayo Memorial Building, 420 Delaware St., S. E., Minneapolis, Minnesota 55455. 0003-9173-78-0500—0475$00.85 © Ar
475
Division of Surgical Pathology, Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, Minnesota
who therefore adopted the name "myospherulosis" for their own cases. It is clear from their description that the nature of these structures puzzled them a great deal. The possibility that they represented "some type of fungus" seemed to attract them the most, and this was reflected in the descriptive terminology they employed, such as fungal-like and sporangium-like. They compared the morphologic features of their cases with those of rhinosporidiosis and coccidioidomyosis, and established that despite some superficial similarities, there were substantial differences among them. Furthermore, the negative staining reaction of the structures in question with Gomori's methenamine silver (GMS) and periodic acid-Schiff (PAS) and their uniform failure to grow in cultures did not support a fungal etiology. They concluded that the nature of the structures was unsettled, but suggested that they had been introduced by the antibiotic ointments. In that regard, they pointed out that these medications are not required to be sterilized (implying that they might be contaminated by the organisms) and recommended a temporary ban of antibiotic ointments or petrolatum-based gauze as packing material. 5 The notable occurrence of 16 patients with this disease in one medical center in a relatively short time was emphasized, as well as the dismay of the authors upon finding that every example in their series had been overlooked by the surgical pathologist who had initially examined the tissue sections. We recently had the opportunity to study a case of this condition occurring in the maxillary sinus of a child. This has given us the opportunity to evaluate these mysterious formations, to perform a series of special staining reactions on them, and to carry out a very simple experiment, which we believe throws some light on their nature. Report of a Case An 8-year-old boy was seen at a hospital in Omaha, Nebraska, because of a mass in the lower aspect of the left orbit. Roentcan Society of Clinical Pathologists
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 7, 2016
Rosai, Juan: The nature of myospherulosis of the upper respiratory tract. Am J Clin Pathol 69: 475-481, 1978. A case of myospherulosis of paranasal sinuses similar in appearance to those recently seen in St. Louis, Missouri, was examined to ascertain the nature of the sac-like structures seen in this disease. The morphologic features of these formations and their positivity with stains for hemoglobin, peroxidase, and lipofuscin strongly suggest that they represent collections of erythrocytes altered by a foreign substance. This interpretation was confirmed by experimental production of these structures by the action of tetracycline ointment on a pure preparation of human erythrocytes. (Key words: Myospherulosis; Erythrocytes.)
ROSAI
476
genograms of the region demonstrated changes consistent with a soft-tissue tumor, with extension into the maxillary bone. An exploration was performed and biopsies of the maxillary sinus and bone were taken. At the conclusion of the procedure, the cavity was packed with gauze impregnated with 3% tetracycline ointment (Achromycin*). Following a pathologic diagnosis of fibromatosis, a radical maxillectomy was performed. The interval between the biopsy and the operation was 13 days. A local recurrence developed in the skin of the cheek overlying the area of the previous excision 5 months after the surgical procedure and was excised. The patient remains free of disease 5 months later.
Material and Methods
Results Sections from the first biopsy and the late skin recurrence showed features of fibromatosis. Sections from the maxillectomy specimen stained with hematoxylineosin showed, in addition to the fibromatosis, numerous tissue spaces of large size. Some were surrounded by a fibrous stroma and others by a granulomatous reaction composed of histiocytes and foreign-body giant cells. A marked lymphoplasmacytic inflammatory response was present beneath the respiratory epithelium. Some of the spaces were empty; others contained a variety of colorless amorphous debris with the appearance of foreign bodies, as well as numerous sac-
like structures identical to those described in the previously mentioned articles, as judged from their description and illustrations (Fig. \A). They ranged in size from 20 to 100 jum. The majority were lying free in the spaces; a few were attached to the wall and gave the impression of actually arising from it. Their shape was round or oval and their color varied from pale eosinophilic to dark brown. Most of the structures were various shades of brown; in some this color was so intense and dark as to almost obscure the inner structure. Finally, some formations (usually of smaller sizes than the preceding ones), were uniformly dark brown, without discernible inner structures; these corresponded to the "brown bodies" of the St. Louis authors. 5 Close examination of the sac-like formations showed that they had in their interiors variable numbers of spherules, measuring 5 /tm on the average (Fig. IB). In contrast to the extreme variations in size of the sacs, these spherules were relatively uniform in size, the range being from 4 to 7 /j,m. Their numbers per sac ranged from 2 or 3 to more than a hundred. Most of these spherules had a round configuration, but others were collapsed and adopted a concave shape; indentations and other irregularities of the membrane were common. Their content was invariably light, structures resembling nuclei being uniformly absent. The spherules staining a pale eosinophilic color had a thin, smooth membrane quite reminiscent of that of erythrocytes; those that had a brown color were noticeably thicker and coarser, with an irregular granularity on the outside. These findings suggested the deposition of a foreign material onto a pre-existing biologic structure. The appearance of the wall of the sac recapitulated that of the inner structures, in terms of both thickness and staining reactions. When cut tangentially, a coarse granularity could be appreciated. Examination of unstained sections mounted in glycerin-water under high contrast conditions (obtained with a dark field or by simply lowering the condenser in a standard bright field) revealed that some of the sac-like structures had a thin, sharply defined membrane, whereas others had a distinct double-layered membrane measuring approximately 1 pm thick (Fig. 1C). The appearance was remarkably similar to that of the erythrocytes present elsewhere in the section. Periodic acid-Schiff, Mayer's mucicarmine, and Giemsa stains were uniformly negative. Gomori's methenamine silver was largely unreactive; however, a few of the structures showed a definite membrane stain of the spherules; in appearance and intensity of the reaction they were similar to occasional clumps of erythrocytes present elsewhere in the specimen. The iron stain was negative for the sacs and their con-
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Microscopic slides from the radical maxillectomy specimen were stained with hematoxylin-eosin, periodic acid-Schiff (PAS), Gomori's methenamine silver (GMS), Mayer's mucicarmine, Giemsa, iron (Perls' stain), lipofuscin (Schmorl's stain), hemoglobin (Okajima's stain) and peroxidase (diaminobenzidine stain). 3 ' 6 ' 10 In an attempt to reproduce the structures seen in the tissues, the following experiment was performed: the inside bottom of a series of test tubes was coated with a thick layer of 3% tetracycline ointment (Achromycin®); 0.5 ml of a fresh preparation of packed human erythrocytes was added to each tube and the preparations were incubated in a water bath at 37 C, with or without shaking, for 1, 2 and 5 days. Following incubation, a portion of the mixture was smeared in a glass slide, fixed in absolute ethanol, and stained with hematoxylin-eosin. Another portion was admixed with thrombin, 9 dehydrated, embedded in paraffiin, sectioned, and stained with the same battery of reagents used for the operative specimen. Control preparations, which were processed and stained in an identical fashion, included (1) 3% tetracycline ointment taken out directly from the dispenser tube; (2) 3% tetracycline ointment after incubation at 37 C for the same periods as above; (3) fresh packed human erythrocytes after incubation at 37 C for the same period as above.
A.J.C.P. . May 1978
NATURE OF MYOSPHERULOSIS
Vol. 69 • No. 5
Microscopic examination of the smears and microscopic slides prepared from the mixture of tetracycline ointment and human erythrocytes showed the presence, among collections of unremarkable erythrocytes and clumps of amorphous colorless material, of structures recapitulating in most respects the appearance of those seen in the maxillary sinus (Fig. 2). This applied to their size, shape, morphology and all of the staining reactions mentioned above. The only major difference was that, in sections stained with hematoxylin-eosin, most of these structures were eosinophilic; only a few showed the brown color seen so prominently in the operative specimen. The wall was thin and smooth in some spherules, and thick and coarsely granular in others. Again, their appearance was strikingly reminiscent of that seen in the human material. This observation particularly applied to the unstained sections examined under high-contrast conditions. These structures were present in all the experiments, regardless of the time and type of the incubation and method of preparation of the specimen, although they were seen in greater number and better preservation in the 24-hour sample. Sections and smears from the erythrocyte controls (without tetracycline ointment) showed only erythrocytes in a good state of preservation, without clumping or brown discoloration. Sections and smears from the tetracycline ointment (without erythrocytes) showed only an amorphous colorless material. Discussion We were just as puzzled as our St. Louis colleagues about the nature of the sac-like structures when we detected them in the case here described. Our first
impression, like theirs, was that they represented some type of fungus. Yet, we had the same inability to relate them to any known type of pathogenic fungus and to explain, if they were indeed fungi, why they would not stain with the PAS reaction or grow in the appropriate cultures. We also considered the possibility of pollen grains or other vegetable matter, but regarded it as unlikely in view of their morphology and alleged negative staining reaction to cellulose. 5 We also looked for help outside our department and showed the slides to microbiologists, parasitologists and experts in microscopic identification of particles. None of them could identify the structures, but we found amusing and eventually intriguing their recurrent comment that the spherules within the sac resembled erythrocytes. We had made that observation ourselves, but not elaborated further on it in view of the brown color of the spherules and the observation that they were contained within a sac. The fact that several other observers were struck by that similarity led us, finally, to ask ourselves this very simple question: is it possible that the reason the spherules resemble erythrocytes so much is simply that they are erythrocytes, altered by the action of the antibiotic ointment? That certainly would have explained their negativity with PAS and other special stains, lack of nuclear structure, and failure to grow in culture media. Even the equivocal stain seen with Gomori's methenamine silver stain could be explained, inasmuch as some of the erythrocytes present elsewhere in the sections reacted similarly. We then proceeded to perform the abovedescribed stains and experiment, the results of which strongly suggest that this is indeed the case. The unequivocal positivity of most of the structures with Okajima's stain gave the first strong support to this hypothesis, since this reaction is regarded as specific for hemoglobin.10 That other structures did not stain with this procedure may be interpreted as the result of leakage of hemoglobin in severely damaged cells. The strong positive reactions obtained with the peroxidase and lipofuscin stains strengthened this impression. However, the most convincing evidence for this interpretation was that we were able to reproduce the structures by the action of the antibiotic ointment on a pure preparation of human erythrocytes. Close examination of the many structures formed in this experiment gave a clue as to their genesis. The affected erythrocytes clumped in groups of various sizes; most of them seemed to lose their biconcave shape in the process, as suggested by the fact that most of them had a round shape in a section in which they were expected to be randomly oriented. A spectrum was found, ranging from spherules with a thin eosinophilic membrane to others with a brown thick membrane containing irregular deposits
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tents, but stained strongly some coarse clumps of amorphous material present in the cytoplasm of multinucleated giant cells. With Okajima's hemoglobin stain, most of the saclike structures showed an unequivocal positive reaction, paler but otherwise identical to that seen in the erythrocytes present in the specimen (Fig. ID); only a few of the formations remained negative. The diaminobenzidine reaction for peroxidase was strongly positive in all the sac-like structures and similar in every respect to that seen in adjacent erythrocytes (Fig. IE). Whenever one of the two above-mentioned reactions was positive in a given structure, this was true both for the spherules and for the sac that contained them. With Schmorl's lipofuscin stain, some of the structures stained pale blue, similar to erythrocytes; other formations (especially the "brown bodies") showed a much stronger, dark blue positive reaction in the interior of the sac between the spherules (Fig. I F ) .
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FIG. 1. Myospherulosis of paranasal sinus. A, several sac-like structures containing numerous spherules can be seen in a large tissue space, one of them attached to the wall. Hematoxylin and eosin. x500. B, higher magnification of two sac-like structures. The lower one is thick-walled and dark brown; the upper one is thick-walled, pale, and eosinophilic. Hematoxylin and eosin. x 1,250. C, unstained section, x 1,250. D, Okajima's hemoglobin stain, x 1,250. E, diaminobenzidine stain, x 1,250. F, Schmorl's stain, x 1,250. The ill-defined clear round areas within the sac correspond to the spherules.
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FIG. 2. Sac-like structures formed by the action of tetracycline ointment on a pure preparation of human erythrocytes. A, hematoxylin andeosin. x500. ,8, hematoxylin andeosin. x 1,250. C, unstained section, x 1,250. D, Okajima's hemoglobin stain, x 1,250. £ , diaminobenzidine stain, x 1,250. F, Schmorl's stain. x750.
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480
ROSAI
A.J.C.P. • May 1978
present in the sections were empty, and thus resembled the spaces seen in other conditions associated with accumulation of abnormal lipids, such as sclerosing lipogranuloma. Even when the sac-like structures were present, they occupied only a small area in the cavity. This suggests that the spaces were originally filled with the oily material used as the carrier for the antibiotic, which had been removed during the tissue processing. This by itself is a possibility worth investigating further. The final consideration involves the possibility that these conclusions could also apply to the African cases. Since we have not had the opportunity to examine them personally or to perform special stains on them we cannot establish it. There are, however, some features that suggest that this might well be the case. Their morphology is said to be identical to that in the American cases, 5 except for their color, which might be a function of the specific agent inducing the change. Hutt and colleagues, 4 in their microscopic description of the cases from Uganda, repeatedly commented upon size and other similarities between the spherules and erythrocytes. They even suggested that the disease might have been produced by the injection of foreign substances, in view of the fact that most of them appeared at "popular injection sites." It is hoped that individuals having access to this material will examine this proposition by performing the same staining reactions that we have applied to the American disease described by Kyriakos.
We did not address ourselves to the question of which component in the antibiotic ointment is responsible for this phenomenon. We suspect it is the petrolatum base rather than the antibiotic, but will let others (perhaps the manufacturers of the medicament) work out the specifics of this biologically intriguing phenomenon. The evidence provided in this article in the sense that the structures in question are not microorganisms of any kind, but rather badly battered erythrocytes, answers the practical questions raised in previous publications about the reasons for the mysterious appearance of an African disease in the American midwest, the possible contamination of antibiotics with microorganisms (for which we found no evidence), and the suggestion of banning their use (which may not be necessary after all). Actually, that these structures have been overlooked until now does not seem, in retrospect, to have been of grave consequence. The fact still remains that an inflammatory reaction of foreign-body type was often present around the formations. We suspect that this reaction was directed not against the clumped erythrocytes but against the foreign material introduced with the antibiotic. In this regard, it may be significant that many of the large tissue spaces
Acknowledgments. The following individuals, all but one from Minnesota, examined the microscopic slides and/or were otherwise helpful in the performance of this study: Dr. Donna Blazevic. Mr. Ray G. Bowman, Dr. Richard Brunning, Mr. Edward Cushing, Dr. Louis P. Dehner, Mr. Barton P. Epstein. Dr. Richard D. Estensen, Ms. Barbara Evans, Ms. Lilian Huehnert. Mr. James Jagodzinski, Dr. Donald Larson, Dr. Jeffrey McCullough. Dr. Edward B. Price (Omaha, Nebraska). Dr. Walter Runge. Ms. Joanne Samuelson, Dr. Arthur Sanders, Mr. James Tucker. ADDENDUM
Following the submission of this manuscript, additional data were obtained which tend to substantiate the opinions expressed in the text. Paraffin blocks of two cases of myospherulosis from Uganda were obtained through the kindness of Dr. Alexander C. Templeton, The St. Vincent Hospital, Worcester, Mass., Prof. M. S. R. Hutt, Dept. of Morbid Anatomy, St. Thomas Medical School, London, England, and Prof. R. Owor, Dept. of Pathology, Makerere University, Kampala, Uganda. Examination of hematoxylin-eosin sections confirmed the original descriptions as well as the marked similarity with the American cases, except for the color of the formations, which were pale eosinophilic instead of brown. The Okajima's hemoglobin stain and the diaminobenzidine reactions were both strongly positive in many of the formations, thus indicating that the "myospherules" of the African cases are also made up of red blood cells.
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on the outside; this was interpreted as the result of a progressive alteration due to increasing deposition of a foreign material on them. The brown discoloration was attributed to the accumulation of decomposition products of hemoglobin rather than to a direct impregnation by the antibiotic ointment, which was colorless in smears and sections. The positivity with the Schmorl's reaction for lipofuscin would seem to support this interpretation. That there was a greater proportion of brown structures in the specimen than in the experiment could be a function of time, which was 13 days in the former and only 5 days or less in the experiment. It was difficult to decide whether the envelope was made up by erythrocytic membranes or exclusively by some foreign material. It seems difficult to believe that erythrocytic membranes can rearrange themselves to make a continuous sphere 100 urn in diameter or more. Yet, the positive reaction of this envelope with the hemoglobin, lipofuscin and peroxidase stain and its alleged morphologic similarity to the membrane of the spherule by electron microscopy 1 suggest that this is indeed the case. Incidentally, the ultrastructural description provided by De Schryver-Kecskemeti and Kyriakos 1 is consistent with our interpretation. Even if the authors do not clearly state it, it is obvious from their electron micrographs that the walls of the spherules and envelope are not normal biological membranes of any sort and that, if they are membranes at all, their inner structure has been totally obscured by the deposition of an extraneous material.
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NATURE OF MYOSPHERULOSIS
481 References
1. De Schryver-Kecskemeti K, Kyriakos M: Myospherulosis. An electron-microscopic study of a human case. Am J Clin Pathol 67:555-561. 1977 2. De Schryver-Kecskemeti K, Kyriakos M: The induction of human myospherulosis in experimental animals. Am J Pathol 87:33-40, 1977 3. Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal convoluted tubules of the mouse kidney: Ultrastructural cytochemistry by anew technique. J HistochemCytochem 14:291-302, 1966 4. Hutt MSR, Fernandes JJ, Templeton AC: Myospherulosis (subcutaneous spherulocystic disease). Trans R Soc Trop Med Hyg 65:182-188. 1971 5. Kyriakos M: Myospherulosis of the paranasal sinuses, nose and middle ear. A possible iatrogenic disease. Am J Clin Pathol 67:118-130, 1977 6. Luna LG (editor): Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Third edition, New York, McGraw-Hill, 1968 7. McClatchie S, Bremner AD: Unusual subcutaneous swellings in African patients. East Afr Med J 46:625-633. 1969 8. McClatchie S, Warambo MW, Bremner AD: Myospherulosis. A previously unreported disease? Am J Clin Pathol 51:699704, 1969 9. Naylor B, Smith MJ: Techniques of cytopathology. Ann Arbor, University of Michigan, 1970 10. Okajima K: On the selective staining of the erythrocyte. Anat Rec 11:295-296, 1916
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An additional feature of interest in one of these cases was the presence of sacs similar in size and shape to those with the "spherules" but containing instead degenerated neutrophils. Prof. Hutt commented that he has recently seen a "definite case in a senior doctor working in the States. This was a lesion in the buttock and it was of interest that he had, at one time, injected himself with a rather dubious antibody when he was traveling in a country with poor medical care." Dr. Stephan A. Szumski, of Lederle Laboratories, Pearl River, N.Y., made the following comment in regard to the brown color seen in the American cases, that we had interpreted as probably resulting from the accumulation of decomposition products of hemoglobin: "The decomposition product in all probability is acid hematin, the same product produced by the action of dilute hydrochloric acid on erythrocytes. It was the basis for the old test used to determine hemoglobin levels in the blood. In that test, a measured amount of blood was added to dilute HC1 and the brown color (acid hematin) produced was measured by comparison with a tinted glass standard. We would expect that this property of HC1 would be retained by the tetracycline hydrochloride in the ointment. The latter is a salt of a weak base, tetracycline, and a strong acid, HCI, the pH is approximately 2.0."
