VHA Diagnostic Electron Microscopy Program
Examples of Diagnostic Scanning EM (SEM)/Energy Dispersive X-ray Analysis (EDX) Cases
Contributed by Durham VAMC EM Program
Amosite and chrysotile asbestos fibers:
This 38 year old New York City fire fighter was at ground zero for 13 days following the September 11, 2001 terrorist attack. He was working 16 hour days, and presented with shortness of breath. Chest x-ray and CT scan showed alveolar infiltrates. Bronchoalveolar lavage was performed, which showed a striking eosinophilia. The patient was treated with oxygen and steroids, and steadily improved over the next few weeks.
Bronchoalveolar lavage macrophages were sent in formaldehyde to our laboratory for analysis of particulate and fiber content by SEM and EDXA. We identified both amosite and chrysotile asbestos fibers. One chrysolite fiber was 70 microns long but only 0.2 micron wide. Other particulates identified included degraded fibrous glass, coal fly ash, silica, and various silicates. The source of the asbestos and other particulates most likely was the dust released from the collapse of the World Trade Centers, which had been insulated with asbestos many years before at the time of their construction.
REFERENCE: Rom, W.N., Weiden, M., Garcia, R., Yie, T.A., Vathesatogkit, P., Tse, D.B., McGuinness, G., Roggli. V.L.. and Prezant, D.: Acute eosinophilic pneumonia in a New York City firefighter exposed to World Trade Center dust. Am. J. Respir. Crit. Care Med. 166:797-800,2002.
Figs 1 and 2 are the SEM image and EDXA spectrum of a chrysotile fiber. Figure 3 is a degraded fibrous glass particle, and Figure 4 a coal fly ash particle. Figs 5 and 6 are the SEM image and EDXA spectrum of an amosite fiber.
Figure 1 Figure 2 Figure 3 Figure 4
Figure 5 Figure 6
Foreign body granulomas:
This 24 year old woman with sickle cell anemia had received oxycontin for pain related to sickle cell crisis. At autopsy, there were numerous foreign body granulomas associated with small arterioles and alveolar septal walls. Polarization microscopy showed birefringent material which had a platy appearance. The material stained positive for methenamine silver and Congo red. Intravenous drug abuse was suspected.
Scanning electron microscopy was used to identify the foreign body granulomas within paraffin sections of lung parenchyma. The material was very weakly back-scatter positive, indicating a density approximately the same as that of the organic matrix of the lung. EDXA spectra showed carbon and oxygen, but no higher atomic number elements. The findings indicated an organic foreign material that was compatible with microcrystalline cellulose. In combination with the light microscopic and histochemical results, the findings were diagnostic.
Microcrystalline cellulose is filler material used in tablets destined for oral consumption. Some individuals crush the tablets, suspend them in water, and inject them intravenously for a `rush'. The foreign material is of course insoluble and lodges in the pulmonary vascular bed. This can result in fatal pulmonary hypertension. We are seeing this form of IV drug abuse more and more frequently, whereas intravenous talcosis seems to be distinctly less common. Oxycontin contains microcrystalline cellulose as the filler material. According to Newsweek, this is becoming the major drug of abuse in the United States today.
Microscopic: H and E stained sections show pulmonary parenchyma with multiple intravascular granulomas. These granulomas contain numerous plate-like birefringent particulates, which stain strongly positive with GMS and weekly positive with PAS. Secondary electron images readily identified granulomas with foreign material (Figure 1). The foreign material showed similar backscattered contrast to that of the surrounding lung parenchyma (Figure 2). Analysis of the foreign material by means of EDXA disclosed peaks for carbon and oxygen, but no inorganic material (Figure 3). The histologic, histochemical, and ultra structural features of this material are consistent with microcrystalline cellulose.
Fig1SEI - Fig2BEI Fig3 EDXA Spectrum
Reference: Thomashefski, J.F., Hirsch, C.S., Jolly, P.N.: Microcrystalline Cellulose Pulmonary Embolism and Granulomatosis: A Complication of Illicit Intravenous Injection of Pentazocine Tablets. Arch Pathol Lab Med 105: 89, 1981.
Scrotal Stone:
A specimen was referred from a mid 40's old male veteran from another VA Medical Center and identified as a scrotal stone. This was analyzed by scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDXA) and found to be a urinary stone consisting of calcium, magnesium, and phosphorus (Figures 1-6).
A literature review showed that scrotal stones typically arise from a bladder hernia or diverticulum that contains a bladder stone. This is obviously important in terms of patient management, since the removal of the stone from the scrotum could potentially injure the bladder wall. Also, it is important in terms of identifying the type of urinary stone and managing the patient appropriately.
This case also illustrates the strengths and weaknesses of SEM/EDXA vs. x-ray crystallography in stone identification. X-ray crystallography missed the predominantly calcium nidus of the stone which had crystalline features typical for calcium oxalate. On the other hand, SEM/EDXA identified the magnesium and phosphorus components as struvite (magnesium ammonium phosphate), whereas x-ray crystallography identified this component as newberyite (magnesium phosphate). Of interest, our new EDXA spectrometer could distinguish between these two forms since it will detect nitrogen (a component of ammonium).
By light microscopy, an elongate plate-like series of crystals is visible at the center of the cleaved surface (Figure 1). Organized around the central area as concentric layers are more loosely aggregated crystals of different shapes. By secondary electron imaging (Figures 2-4), these crystals are bipyramidal, or in some cases, finely granular spheres. In addition are trapezoidal crystals which are large relative to the other components. Energy dispersive x-ray spectra from five representative regions of the surface contain peaks for phosphorus and calcium, magnesium and phosphorus, or all three elements together (Figures 5-6). Small peaks also occur in some regions for sodium and potassium.
Figure 1 Figure 2 Figure 3 Figure 4
Figure 5 Figure 6
Interpretation of Structural and Analytical Results:
The presence of magnesium, calcium, and phosphorus, together with characteristic crystal shapes, are consistent with the stone being composed of calcium oxalate monohydrate (whewellite), calcium phosphate (apatite), and magnesium ammonium phosphate (struvite). The nidus is composed of calcium oxalate monohydrate as evidenced by the flat, tabular crystals located centrally. Calcium phosphate crystals and magnesium ammonium phosphate crystals occur in the periphery of the stone beyond the nidus. Calcium oxalate monohydrate occurs in a pathophysiological setting of hyperoxaluria: it is frequently associated with Calcium phosphate as a result of intermittent infections with non-urease producing bacteria. In contrast, magnesium ammonium phosphate results from infections with urease-splitting bacteria; it is almost always associated with calcium phosphate since both require an alkaline environment for precipitation to occur. Additional confirmation of these crystalline types will be obtained by x-ray diffraction on a portion of the stone which has been ground to a powder; a supplement to the report will be added when those studies are completed. In this case, the composition and morphology are more consistent with a bladder stone that ruptured through the bladder wall into the scrotal sac. Another possibility is scrotal hernia of the bladder with bladder calculi.
References;
Gayet; R. , Scrotal hernia of the bladder in a patient with prostatic adenonoma and bladder calculi. J Urol Nephrol (Paris) 73: 288; 1967
Postma M.P., Smith, R. Scrotal cystocele with bladder calculi (case report). Am J. Roentgenol 147:287; 1986
Alveolar proteinosis:
A 52 year old man presented with dyspnea, and was found to have bilateral air-space disease on chest x-ray. An open lung biopsy showed the typical features of pulmonary alveolar proteinosis. He had a history of working with indium containing dust in a hydrogen furnace. The case was referred to try to determine whether the exposure to indium had any relation to the patient's alveolar proteinosis.
We received a paraffin block and an epon block prepared from the open lung biopsy. A section of the block was placed on a carbon planchet, rotary carbon coated, and examined in a scanning electron microscope (SEM) equipped with an energy dispersive x-ray detector (EDXA). By back-scattered electron imaging, dense particles were readily identified (Figure 1), and EDXA showed a composition of indium (In) (Figure 2). These results were transmitted to the referring pathologist on March 2, 2001.
Because the alveoli were completely consolidated by the proteinaceous material, it was difficult to localize the particles by SEM. Therefore, thin sections were prepared from the Epon block and examined by scanning transmission electron microscopy (STEM) and EDXA. Electron dense particles were readily identified, both within the alveolar lipoproteinosis debris (Figure 3) and within the cytoplasm of alveolar Type II cells (Figure 4). The particulates were again identified as In (Figure 5).
Alveolar proteinosis has been described in association with exposure to excessively high levels of several types of fine dust particles, including silica, aluminum, and titanium. This case represents the first association between pulmonary alveolar proteinosis and exposure to indium.
Figure 1 Figure 2 Figure 3 Figure 4
Figure 5
Apatite/weddellite stone by SEM/EDXA:
A 49 year old man submitted 'gravel' passed in the urine to another VA facility, and this material was submitted to our VA Medical Center for analysis. We are presently examining all urinary stones at our facility with a combination of scanning electron microscopy and energy dispersive x-ray analysis. This combination provides information regarding the crystal habit of the stone as well as its elemental composition. Together, these two pieces of information allow accurate identification of 90% or more of the stones that we receive for analysis. This accuracy rate was determined by comparing our findings with those of x-ray diffraction, a widely accepted technique in the field, on the same samples. Furthermore, we learned that SEM/EDXA often detects minor components of the stone that are missed by conventional x-ray diffraction.
In the present case, the identification of granular or spherical morphology in the majority of the stone along with peaks for calcium and phosphorus (Figures 1-4) indicate that the stone consists primarily of calcium phosphate (apatite). In addition, the presence of typical bipyramidal crystals (Figure 3) indicates that there is also a component of calcium oxalate dihydrate (weddellite). Such stones typically occur in the setting of hypercalciuria and/or primary hyperparathyroidism.
Figure 1 Figure 2 Figure 3 Figure 4
Foreign bodies in liver tissue matrix and in Kupffer cells:
This 53 year old woman had bilateral hip replacements and a liver biopsy for other reasons. Granular material was identified within Kupffer cells. The consulting pathologist wondered whether this material might not be derived from wear-particles from the hip replacements.
A section was prepared from a submitted paraffin block and examined by scanning electron microscopy and energy dispersive x-ray analysis. The particulate material within Kupffer cells was located by means of secondary and backscattered electron imaging. EDXA and digital mapping demonstrated the presence of titanium, consistent with wear particles from prosthetic hip joints. The calcium, phosphorus, and sulfur co-localizing with the titanium particles are likely of endogenous origin. We have also identified such wear particles within pelvic lymph nodes in other cases. Such wear particles are more likely to migrate to distant sites when the prosthetic joint becomes loosened from the adjacent bone.
SEI image (Figure 1) revealed a typical liver tissue matrix with cell outlines discernable. Some bright particulate material is visible in this image. By backscatter electron imaging (Figure 2), these same bright regions appeared as very dark, dense particulates. Energy dispersive X-ray spectra (Figure 4) obtained from these particles contained peaks for titanium, phosphorus, calcium, and sulfur. In one area of the specimen, a cell body was observed, measuring approximately 26 x 14 micrometers and containing multiple bright particulate structures in SEI mode. In BEI mode, these same particles were electron dense with EDX spectra from them positive for titanium. Elemental images from this cell are depicted in Figure 3.
Figure 1 Figure 2 Figure 3 Figure 4
Hypersensitivity pneumonitis with CaOx crystals:
This 61 year old woman had an open lung biopsy with features highly suggestive of hypersensitivity pneumonitis (extrinsic allergic alveolitis). However, many of the interstitial giant cells contained birefringent inclusions suggestive of foreign material. This finding raised the question of pneumoconiosis or foreign body granuloma. The paraffin block was submitted for characterization of the birefringent particles by means of scanning electron microscopy and energy dispersive spectrometry.
The particles within giant cells were located by means of secondary and backscattered electron imaging. These particles were then analyzed by EDXA and found to contain calcium, indicating a probable endogenous origin. The SEM/EDXA findings were consistent with my light microscopic impression of calcium oxalate. These crystals are well known to occur in association with certain granulomatous reactions, especially sarcoidosis. Their occurrence in cases of hypersensitivity pneumonitis is less well recognized and resulted in the diagnostic difficulty noted above. The final diagnosis was hypersensitivity pneumonitis with endogenous calcium oxalate inclusions. This opinion was consistent with the patient's history, as she had worked around a grain elevator for the past five years.
Exam by SEM and BEI (Figures 1 -3) demonstrate the presence of strongly back-scatter positive platy particles within the cytoplasm of some multinucleate giant cells. Examination of these particles with energy dispersive spectrometry demonstrates a peak for calcium only, consistent with the histological impression of calcium oxalate. Elemental maps (Figure 4) were also obtained from one cluster of particles and this demonstrates strong positivity for calcium.
Figure 1 Figure 2 Figure 3 Figure 4
Amiodarone pulmonary toxicity:
This 61 year old male Veteran developed respiratory failure after coronary artery by-pass graft procedure. He was found to have severe pulmonary fibrosis at autopsy with superimposed acute respiratory distress syndrome. Numerous foamy macrophages were present in the alveolar spaces adjacent to fibrotic areas. The patient had been placed on amiodarone 1 1/2 years prior to admission. Forty-five days after the CABG procedure, amiodarone levels on autopsy blood were still within the therapeutic range and the levels of an amiodarone metabolite were markedly elevated.
Ultrastructural studies were instituted to try to confirm the clinical impression of amiodarone pulmonary toxicity. Cases of patients on amiodarone developing ARDS following a surgical procedure have rarely been described, and are believed to be due to a synergistic effect between oxygen administered at time of anesthesia and amiodarone accumulated within pulmonary tissues. The mechanism may involve free radical formation. In this case, foamy macrophages were identified ultrastructurally, and myelin figures with features consistent with amidodarone inclusions identified. Electron probe microanalysis demonstrated the presence of rare iodine-containing electron-dense inclusions, further supporting the clinical impression of amiodarone toxicity. Iodine peaks were absent from the adjacent cell cytoplasm.
One micron toluidine blue-stained thick sections show numerous foamy histiocytes within alveolar spaces. Ultrastructurally, these histiocytes contain vacuoles and myelin figures. Areas within some of these cytoplasmic vacuoles (Figure 2) were analyzed using energy dispersive spectrometry. Spectra with peaks characteristic for iodine were obtained. Figure 3 represents spectra from inclusion probe spot. Figure 4 represents spectra from background probe spot.
Fig. 1 Fig. 2 Fig. 3 Fig. 4
