The movat pentachrome stain as a means of identifying microcrystalline cellulose among other particulates found in lung tissue.
The pentachrome stain was originally developed by Movat in 1955 to highlight the various constituents of connective tissue by means of a single histochemical staining procedure. (5) Subsequently, in 1972, Russell introduced a modification of the Movat technique, which greatly reduced the time required for staining and increased the consistency of the staining results. (6) The MMPS is now the preferred pentachrome stain in histology laboratories. The major components of the MMPS, as used in our laboratory, are Verhoeff elastic hematoxylin, sodium thiosulfate, acetic acid, and alcian blue, crocein scarlet-acid fuchsin, and alcoholic saffron solutions. (6,8) It is the saffron component of the MMPS that imparts a yellow hue to the collagen fibers and to the cellulose. In this retrospective study, we evaluated the MMPS as a means of identifying MCC and separating it from other birefringent crystals that are occasionally found in lung tissue specimens.
MATERIALS AND METHODS
Archival, formalin-fixed, paraffin-embedded lung or pleural tissue specimens that contained various birefringent crystals were retrieved from departmental autopsy and surgical pathology files. Crystals encountered within the tissue samples included MCC (3 cases of intravenous drug abuse), crystals consistent with talc (2 cases of intravenous drug abuse, 1 case of talc pleurodesis), mixed silicates (1 case of suspected silicate pneumoconiosis in a rubber worker), and calcium oxalate (1 case of aspergillosis from Aspergillus niger), which were respectively evaluated with the MMPS using routine light microscopy. (8) Crystal identification was confirmed by the morphologic characteristics of the various particles, other histochemical stains, infrared spectroscopy (1 case previously published9), and known cellulose controls. The 2 cellulose controls consisted of a pharmaceutical tablet that contained MCC, and cellulose paper insulation material, each of which was immersed in 10% neutral-buffered formalin, compacted, embedded in paraffin, and routinely processed in the histology laboratory as a "tissue" block. The tinctorial patterns among different crystals following staining with MMPS were compared by qualitative assessment.
To further compare the results of MMPS versus other stains traditionally used for tissue identification of MCC, representative study cases containing MCC or talc, and the 2 cases of mixed silicate or oxalate deposition were also stained using Gomori methenamine silver and Congo red stains.
Patient demographics, the structures of particles seen in lung and/or pleural tissue, and the MMPS stain results are summarized in the Table. Subjects 1, 2, and 3 were included in a previously published report. (9) In these 3 subjects, MCC appeared similar to that seen in the control slide of a pharmaceutical tablet, having a rodlike structure, brightly birefringent under polarized light, and staining grey-black with Gomori methenamine silver, orange with Congo red, and dull to bright yellow with MMPS (Figures 1 through 4). Occasional cellulose fibers stained with MMPS had a yellow-green tint or peripheral yellow-green rim. Microcrystalline cellulose was localized within or around pulmonary artery branches, a location that was easily discernable with the MMPS, which stains vascular elastic tissue black.
Birefringent crystals consistent with talc were present in the lungs of 2 subjects suspected of intravenous drug use (subjects 5 and 6) and in the pleura of 1 patient following talc pleurodesis for recurrent pneumothorax (subject 4). Talc appeared as refractile, stacked crystals that were brightly birefringent and stained weakly light blue with MMPS (Figures 5 through 7). Subject 7, a rubber worker with probable exposure to talc and/or other silicates, had numerous talclike particles associated with interstitial fibrosis. These crystals were brightly birefringent, and were either unstained or tinted weakly light blue with MMPS. Case 8 represents a patient with necrotizing Aspergillus niger pneumonia and lung infarction who was found at autopsy to have extensive deposition of brightly birefringent calcium oxalate crystals having diverse shapes, and focally aggregated into rosettes and wheat sheaflike structures (Figures 8 through 10). Calcium oxalate in this case was confirmed by the rubeanic acid stain of Yasue. (10) The MMPS stained the oxalate crystals light sea-green with loss of their birefringent character (Figure 10).
Crospovidone, another pharmaceutical tablet filler material that was also seen in the lungs of subjects 1 through 3, had an irregular, corallike shape; was nonbirefringent; and stained yellow-green to blue-green with MMPS (Figure 11). (9) Cornstarch was also noted in the tablet control slide as refractile polyhedral particles, which were unstained with MMPS and had a characteristic Maltese-cross birefringence when viewed with polarized light.
In comparison to the results obtained with MMPS, GMS stained cellulose fibers gray to black whereas Congo red imparted an orange color to MCC. Neither GMS nor Congo red stained talc, mixed silicates, or oxalates. Following staining with GMS or Congo red, bright birefringence under polarized light was retained for MCC, talc, and mixed silicates; however, birefringence was lost for oxalates following application of the GMS but not the Congo red stain.
The modified Russell Movat pentachrome stain (MMPS) is an exceptional histochemical technique that highlights a number of tissue structural components in a single slide. Traditionally, MMPS has been used in pulmonary or cardiovascular pathology to assess vascular remodeling. It is also excellent, however, for evaluating long-standing, collagenous fibrosis and for distinguishing advanced fibrosis from more recent fibroplasia. In tissue, MMPS highlights elastic fibers as black, collagen as yellow, smooth muscle as light red, and mucopolysaccharides as green-blue. (5,6) In this study, we extend the utility of the MMPS to assessing birefringent crystals and other particulates that might be seen in lung or other tissue. Our findings corroborate rare isolated case reports in which the yellow appearance of MCC in the MMPS is incidentally illustrated. (11) As emphasized in the present study, MMPS is especially useful in distinguishing microcrystalline cellulose from other birefringent particles, such as talc, for which it is often mistaken.
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Microcrystalline cellulose, along with talc, is an important filler material in pharmaceutical tablets. To the trained eye, MCC can be distinguished from talc by its larger size and its rodlike or matchsticklike appearance. The bright birefringence of each material in polarized light, however, can cause confusion. To our knowledge, there is no readily available histochemical stain for talc or other silicates in tissue. Microcrystalline cellulose, on the other hand, retains the staining properties of native wood cellulose and can be highlighted by periodic acid-Schiff (with or without diastase), Congo-red (without apple-green birefringence), and Gomori methenamine-silver stains. The saffron component of MMPS also stains cellulose yellow, whereas the alcian blue component of the MMPS tends to tint talc particles (and probably other silicates) light blue. The water-soluble carotenoid, crocin, is the key compound in saffron that imparts a yellow hue to MCC. Crocin is a digentibiosyl ester of the polyene dicarboxylic acid crocetin.12 Saffron is thought to be a direct dye. The shape and length of the crocin molecule along the cellulose fibers appears to maximize various bonds between the 2 materials.
Staining of talc and/or other silicates can be quite variable as highlighted in case 7 of this series. It is our assumption that the blue coloration of talc in the MMPS does not represent a true chemical staining reaction with the mineral particle, but likely, represents staining of mucopolysaccharides coating the outer surface of the particle in tissue, or perhaps nonspecific electrochemical bonding between stain and mineral particle.
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There are 2 main pathways by which MCC may enter the lung. The first, and most widely recognized, method occurs through the practice of injecting aqueous suspensions of pharmaceutical tablets intravenously to achieve the pharmacologic effect of the active tablet ingredient. Microcrystalline cellulose, along with talc, crospovidone, and other fillers, embolize to the lung, where they obstruct and destroy the pulmonary microvasculature leading to pulmonary hypertension and cor pulmonale. (3) The MMPS also serves to discriminate crospovidone (polyvinyl-Npyrrolidone) from MCC, although this distinction is usually not as crucial because the identification of crospovidone can be readily made based on the corallike shape of the particle, its lack of birefringence, and its deep basophilia with hematoxylin-eosin stain. (9)
The second, and probably more frequent, route of MCC entry into the lung follows aspiration of pharmaceutical tablets from the oral cavity or stomach. (7,13) The dual value of the MMPS is that it not only helps to identify MCC but also serves to localize the material within either the bronchiolar or the vascular compartment through its elastic staining properties, and thereby suggests the pathway by which the MCC gained access to the lung.
As suggested by our study, MMPS may be useful in identifying other crystalline materials in tissue. This provides an advantage from the use of MMPS over other stains for MCC, such as GMS or Congo red, which do not stain talc, mixed silicate, or oxalate crystals. In our study, calcium oxalate crystals in the lung, in a case of aspergillosis from Aspergillus niger, stained light sea-green with MMPS, accompanied by loss of its bright birefringence. Whether this represents a true staining reaction or is another instance of nonspecific staining of a protein within, or coating, the crystal is uncertain. (14) Calcium oxalate is difficult to identify in tissue by histochemical means. Perhaps the most reliable method, and the one used in this study, is the Yasue stain, which applies aqueous silver nitrate and rubeanic acid to color oxalate black. (10) Unfortunately, the Yasue stain is not readily available in most histology laboratories. There is a well-known association between Aspergillus species, especially A. niger, and pulmonary oxalosis. (15) Distinction of calcium oxalate from other birefringent crystals is not usually problematic in the context of Aspergillus hyphae coexistent in the slide. Occasionally, however, especially in small, endoscopic lung biopsies or cytology specimens, oxalate crystals may be present in the absence of associated fungal hyphae. (16,17) In this situation, the distinction from other crystals is important because oxalate crystals may signal the presence of aspergillosis elsewhere in the lung. (16,17) In a recent publication, (18) oxalate was also associated with pulmonary zygomycosis.
The MMPS may also prove to be useful in evaluating crystalline deposits in cases of alleged intravascular crystalline precipitation of hyperalimentation fluid. This is a poorly defined phenomenon, described mainly in a few case reports, in which brightly birefringent crystals, identified as calcium phosphate in one report, precipitate within the vasculature and may embolize to the lung. (19) In each of 2 separately reported patients who were considered to have crystalline deposition of hyperalimentation fluid, published photomicrographs depict rod-shaped birefringent particles highly suspicious of microcrystalline cellulose (in one of these cases, MMPS was noted to stain the particles yellow). (20,21) In another, very similar case, crystals deposited within the lung were shown by histochemical stains and spectroscopic analysis to, in fact, represent MCC. (22) The patient in this report subsequently admitted injecting codeine tablets into his hyperalimentation line. (22) Indwelling vascular ports, unfortunately, provide ready access for illicit, intravenous drug use, including the injection of pharmaceutical tablets. The careful exclusion of tablet filler materials by crystal morphology, histochemical stains, and, if necessary, analytical methods, such as mass spectroscopy, energy-dispersive x-ray analysis, or Raman laser spectroscopy, should be performed before assuming that crystalline precipitation of hyperalimentation fluid has occurred.
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Despite its usefulness, the MMPS is a technically challenging stain to perform. Inconsistency of the staining result may be a problem, especially when the stain is used infrequently, or when performed by an inexperienced histotechnologist. We have occasionally noticed that MCC may stain green or green-yellow if the timing of the saffron or pH and timing of the alcian blue components of the stain are not carefully monitored. Aspirated plant cellulose material, in our experience, may also have a greenish tint rather than the bright yellow color of MCC. Another potential pitfall in the identification of MCC by MMPS is the overinterpretation of tissue collagen, especially the coarse collagen of vascular adventitia or of interstitial fibrosis, for MCC. Both collagen and MCC express about the same intensity of yellow in the MMPS. The distinction is usually not an issue if careful attention is paid to the rodlike structure and associated foreign-body reaction of MCC versus the long, wavy, layered structure of collagen in its normal perivascular or interstitial location. When in doubt, other histochemical stains, such as GMS or Congo red, can be used to support the identification of MCC.
In summary, the modified Russell Movat pentachrome stain, in addition to its time-honored application in evaluating connective tissue, can be very useful in suggesting the identity of particulates, especially birefringent particles like MCC, in lung tissue. Of course, none of the histochemical stains used in this study are specific for the identification of MCC. However, with careful clinico pathologic correlation, detailed attention to the morphology of the crystals, and application of the MMPS (with other supportive stains as necessary), MCC can be identified with a reasonable degree of assurance in histologic sections, without the need for expensive and time-consuming analytical procedures. The MMPS is particularly useful in evaluating embolized pulmonary crystalline and particulate material associated with illicit intravenous drug use.
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(18.) Rassaei N, Shilo K, Lewin-Smith MR, Kalasinsky VF, Klassen-Fischer MK, Franks TJ. Deposition of calcium salts in a case of pulmonary zygomycosis: histopathologic and chemical findings. Hum Pathol. 2009;40(9): 1353-1357.
(19.) Pomerance HH, Rader RE. Crystal formation: a new complication of total parenteral nutrition. Pediatrics. 1973;52(6):864-866.
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Saroj Sigdel, MD; J. Todd Gemind, HT; Joseph F. Tomashefski Jr, MD
Accepted for publication April 13, 2010.
From the Department of Pathology, MetroHealth Medical Center and Case Western Reserve University School of Medicine, Cleveland, Ohio. Dr Sigdel is now with the Department of Pathology, St Mary's Medical Center, Huntington, West Virginia, and Mr Gemind is now with the Cleveland Skin Pathology Lab, Inc, Beachwood, Ohio.
The authors have no relevant financial interest in the products or companies described in this article.
Presented in part at the annual meeting of the United States and Canadian Academy of Pathology, Atlanta, Georgia, February 15, 2006.
Reprints: Joseph F. Tomashefski Jr, MD, Department of Pathology, MetroHealth Medical Center, 2500 MetroHealth Dr, Cleveland, OH 44109 (e-mail: firstname.lastname@example.org).
Demographic Data, Crystal Morphology, and Staining Results Case No. Age, y/Sex Material Clinical Setting 1 40/F MCC IVDA Crospovidone (a) 2 27/M MCC IVDA Crospovidone (a) 3 38/F MCC IVDA Crospovidone (a) 4 40/F Talc Pleurodesis 5 50/M Talc IVDA 6 37/F Talc IVDA 7 73/M Mixed silicates Occupational 8 72/M Oxalate Aspergillosis from Aspergillus niger Tablet control MCC Starch Paper control Cellulose fibers Case No. Structure MMPS Results 1 Rodlike Bright yellow Corallike Yellow-green 2 Rodlike Bright yellow Corallike Yellow-green 3 Rodlike Yellow Corallike Dark-olive green 4 "Stacked plates" Light-blue tinged 5 "Stacked plates" Light-blue tinged 6 "Stacked plates" Light-blue tinged 7 "Stacked plates" Light-blue tinged or unstained 8 "Wheat sheaves" Sea-green, loss of birefringence Tablet control Rodlike Bright yellow Maltese cross Unstained Paper control Rodlike Bright yellow Abbreviations: IVDA, intravenous drug abuse; MCC, microcrystalline cellulose; MMPS, modified Russell Movat pentachrome stain. (a) Particles, nonbirefringent.
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|Author:||Sigdel, Saroj; Gemind, J. Todd; Tomashefski, Joseph F., Jr.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Date:||Feb 1, 2011|
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