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Polystyrene Microspheres as a Specific Marker for the Diagnosis of Aspiration in Hamsters

Institute of Pulmonology and Department of Pathology, Hadassah University Hospital and Hadassah-Hebrew University Medical School, Jerusalem, Israel; and Department of Chemistry, Bar–Ilan University, Ramat-Gan, Israel

Address correspondence to: Dr. A. Avital, Institute of Pulmonology, Hadassah University Hospital, POB 12000, Jerusalem 91120, Israel. E-mail: avital{at}hadassah.org.il

	Abstract

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The diagnosis of recurrent aspiration in young children is problematic because there is no specific gold standard test to be used. In the present work, normal saline or a suspension of white polystyrene microspheres in normal saline was instilled into hamsters' trachea (n = 42), and bronchoalveolar lavage (BAL) cytology, microsphere index (total microspheres/100 macrophages), and lung histology were followed for 90 d. Naive animals (n = 6) had no tracheal instillation. On Days 1, 3, 10, 32, 60, and 90 after tracheal instillation, animals were killed (saline-instilled animals, n = 3; and microsphere-instilled animals, n = 4), and BAL was performed. There was a marked inflammatory response in BAL on Day 1 after tracheal instillation of saline or microsphere suspension. White microspheres were clearly identified within alveolar macrophages in all studied days. Microsphere numbers showed a 50% disappearance rate of 10 d. A mild peribronchial inflammation was noted in lung histology only on Day 1 after instillation. Microspheres were not detected in extrapulmonary organs. We conclude that polystyrene microspheres instilled in hamsters' trachea can be easily identified in BAL macrophages for as long as 3 mo and could potentially be used as a sensitive, specific, and stable marker for the diagnosis of aspiration.

Abbreviations: bronchoalveolar lavage, BAL

	Introduction

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The diagnosis of recurrent aspiration pneumonia in young children is a perpetual challenge because there is no gold standard test sufficiently specific to confirm the diagnosis. Barium swallow with videofluoroscopy and radionuclide milk scintigraphy (milk scan) are neither sensitive nor specific for the diagnosis of aspiration (1, 2). The finding of lipid-laden alveolar macrophages in bronchoalveolar lavage (BAL) was found to be a sensitive but nonspecific test in children and adults (3, 4). Recently, Elidemir and colleagues (5) described a novel diagnostic method for aspiration in a murine model. They stained milk proteins (-lactalbumin and ß-lactoglobulin) in alveolar macrophages by an immunocytochemical method. They found that their method is very sensitive and very specific as compared with Oil-Red O staining, but the stainings were positive only for the 3–4 d after induced aspiration. Aspiration can occur from above, as in children with neurologic impairment, but it may occur secondary to gastro-esophageal reflux and may occur only during the massive episodes and not necessarily every day. It is therefore important to have a sensitive and specific marker that could help diagnose aspiration that may have occurred days or weeks previously. This marker should be made from an inert nonharming material that can be given orally with food, is not produced endogenously, can easily be "swallowed" by alveolar macrophages, and can be identified in BAL alveolar macrophages for a substantial period of time. In a recent study (6), we used activated charcoal particles mixed with milk or normal saline and performed tracheal instillation in hamsters. Charcoal particles were easily identified in BAL for the 3 mo after a single instillation, although hypercellularity was found in BAL after 3 mo, and it was attributed to the fact that some of the particles were large and may have induced airway inflammation. In the present study, we performed tracheal instillation of small (2.1 µm) and uniform polystyrene microspheres suspended in a saline solution in hamsters; compared it with tracheal instillation of normal saline alone; and followed for BAL cytology, microsphere index, and lung histology during the following 3 mo.

	Materials and Methods

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Animals
Fifty-seven female Syrian hamsters (Mesocricetus auratus; Harlan Sprague Dawley, Indianapolis, IN) 9–12 wk old and weighing 100–140 g were used for the experiments. The study was approved by the local Institutional Animal Care and Use Committee.

Six hamsters were used as naive controls (without tracheal instillation). Forty-two hamsters had 0.1-ml tracheal instillation of sterile normal saline (n = 18) or white polystyrene microspheres (n = 24) of uniform size (2.1 µm). On Days 1, 3, 10, 32, 60, and 90 after instillation, the animals were killed (saline-instilled animals, n = 3; microsphere-instilled animals, n = 4), and BAL was performed. Nine additional hamsters had tracheal instillation of black polystyrene microspheres to visualize the microspheres within extra-pulmonary organs, including perihilar lymph nodes. They underwent BAL and were killed on Days 10 (n = 3), 20 (n = 3), and 30 (n = 3) after instillation.

Polystyrene Microspheres
The white polystyrene microspheres were formed by dispersion polymerization of styrene in alcohol as done previously and had a diameter of 2.1 ± 0.1 µm (mean ± SD) (7). Black polystyrene microspheres were formed using Sudan-black dye introduced by a single step swelling procedure as recently published (8). All microspheres were efficiently suspended (10 mg/ml) within sterile normal saline by sonification as shown by light microscopy.

Anesthesia
A mixture of ketamine HCl (50 mg/ml, 10 ml) and dehydrobenzperidol (2.5 mg/ml, 2 ml) was administered by intraperitoneal injection; 0.25 ml was administered before tracheal instillation, and 0.4 ml was administered before exsanguination and BAL.

Tracheal Instillation
Direct intubation was performed with a blunted metal needle. A few ventilations with a small ambu bag were given to ascertain intratracheal position of the needle by confirming chest movement. Normal saline or microsphere suspension (0.1 ml) was slowly instilled into the trachea, followed by a few ventilations with the ambu bag to disperse the fluid and to prevent apnea.

BAL
Exsanguination was performed under anesthesia by transection of the abdominal aorta. The trachea was exposed, and a blunted needle covered with a polyethylene cannula was inserted into the trachea. Three aliquots of 5 ml sterile 0.9% saline were injected and withdrawn, with a total recovery of 85.3% ± 0.8%. The fluid was examined for total cell counts, and slides for differential counts were prepared on a Cytospin 3 (Shandon, Cheshire, England) using 100 µL of BAL fluid.

Microsphere Index
The first 100 consecutive intact macrophages viewed were evaluated, and the total number of white microspheres within the 100 macrophages was defined as the microsphere index on each BAL day.

Histology
Lungs from all animals were preserved in formaldehyde for further evaluation, and four slides from both lungs were prepared. Other internal organs (kidneys, liver, spleen, adrenals, pancreas, heart, and brain) from eight hamsters after 90 d of tracheal instillation (five after white microsphere instillation and three after saline instillation) were preserved in formaldehyde for further evaluation. Animals instilled black microspheres had all extrapulmonary organs, including perihilar lymph glands, preserved in formaldehyde for histologic evaluation. Two slides from each extrapulmonary organ were prepared. Hematoxylin-eosin was used for staining. Slides were thoroughly evaluated by two senior pathologists (V.D. and Y.S.).

Statistics
Group data are expressed as mean ± standard error of the mean. Differences between groups were compared using analysis of variance with Bonferroni correction. Differences were taken as significant when P < 0.05.

	Results

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The results of total cell counts, total macrophages, percent neutrophils, and microsphere index were performed on BAL of hamsters instilled with normal saline or white polystyrene microspheres and are presented in Table 1.

Percent Neutrophils
There was a short inflammatory response to tracheal instillation of either saline or microsphere suspension. Percent neutrophils increased similarly and significantly in both groups (P < 0.01 and P < 0.001, respectively) as compared with the naive controls during the first day after tracheal instillation (Table 1). Thereafter, percent neutrophils returned to normal values in both instilled groups with no significant differences between the groups.

Microsphere Index
White polystyrene microspheres were easily identified within alveolar macrophages in all studied days and within neutrophils (12.5%) only on Day 1 after tracheal instillation. No microspheres were found in the BAL of baseline or saline-instilled animals. Results of microsphere index during the days after instillation are presented in Table 1. The rate of decrease in this index showed a logarithmic decay, with a 10-d 50% rate of disappearance. The percentage of macrophages free of microspheres (mcs = 0), or containing 1–5 or > 6 microspheres is illustrated in Figure 1. White and black microspheres within alveolar macrophages (Days 1 and 10, respectively) are shown in Figure 2.

View larger version (16K): [in this window] [in a new window]   Figure 1. Percent macrophages free of microspheres (mcs = 0), with 1–5 microspheres and with 6–10 microspheres along time.

View larger version (80K): [in this window] [in a new window]   Figure 2. White (A) and black (B) microspheres within alveolar macrophages from BAL performed on Days 1 and 10, respectively, after tracheal instillation.

Other Organ Histology
Neither white nor black microspheres were found within perihilar lymph nodes, kidneys, liver, spleen, adrenals, pancreas, heart, or brain of instilled animals, and the histology of those organs was normal.

	Discussion

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In this study we instilled a suspension of white polystyrene microspheres into hamsters' trachea and could easily identify the uniform microspheres within alveolar macrophages during the next 3 mo after a single tracheal instillation. The microspheres were identified on Day 1 within alveolar neutrophils and macrophages and in the subsequent days only within the macrophages.

The half-life disappearance of the microspheres was 10 d; however, the microspheres were easily identified until Day 90 (end of study) after a single tracheal instillation. The fact that this marker can be identified in alveolar macrophages for at least 3 mo after tracheal instillation allows its practical use as a very sensitive, specific, and stable marker for the detection of aspiration. Its sensitivity was 100% because the microspheres were detected in all the BALs performed, and its specificity was 100% because the microspheres were not found in naive or saline-instilled animals. This high sensitivity and specificity occurred on all studied days, making the use of this marker in humans promising. There are a few limitations in applying this technique in humans. Most patients who aspirate may not do so with every feeding or every day, so we may need to include a few meals with microspheres every day for approximately 1 week before we perform bronchoscopy and BAL on children with suspected aspiration syndromes. Another important point is that we do not perform a whole lung lavage in children like in our animal model, and therefore we may perform BAL from a lobe or lung that had not aspirated food with our marker. In humans, although we expect to reach a high specificity, the sensitivity of this test will probably be lower than that obtained in our animal model.

The inflammatory response seen in BAL on Day 1 after tracheal instillation was similar in the saline and microsphere-instilled animals. The maximal cellular response, caused mainly by a neutrophil increase, was detected during the first day and returned to normal thereafter. There was a moderate increase in total macrophage count compared with a two- to three-fold greater increase in total macrophages found in BAL of hamsters who had milk or milk-charcoal instillation (6). This suggests that, in hamsters, the polystyrene microspheres behave as an inert material and do not induce a sustained inflammatory response in the airways or in the lung parenchyma.

The main known toxicity of polystyrene is related to apparent inhalation or absorption by workers exposed to the constituents of polystyrene during its manufacture. Polystyrene is a polymer of styrene; it is a monoaromatic hydrocarbon and a colorless liquid at room temperature. The main use of styrene is in the production of polystyrene, foam materials, waxes, automobile tires, plastics, varnishes, and synthetic rubber products (9). Employees of polystyrene laboratories were found to have an increased incidence of pancreatic, colon, and lung cancer (10, 11).

In medicine, polystyrene microspheres have been used in the treatment of hepatocellular carcinoma, based on the principle that the vascular bed of many solid tumors is hyperpermeable to macromolecules. This increased vascular permeability, together with an ineffective lymphatic drainage, causes the selective accumulation of the macromolecules within the tumor. This enhanced permeability and retention effect is used to increase the potency of cytotoxic drugs. A conjugate of poly(styrene-comaleic acid) and neocarzinostatin (cytotoxic), called styrene-maleic/anhydride-neocarzinostatin, is in clinical use in Japan for the treatment of hepatoma (12–14) and pleural or peritoneal cancer (15).

Antibody-conjugated beads (immunobeads) have been used to isolate tumor cells from blood, bone marrow, ascitic/pleural fluids, and enzyme-digested tissue biopsies (16).

Labeled polystyrene microspheres given by inhalation have also been used in human studies for measurement of mucociliary clearance rates and airway resistance (17–22).

In our study, there was no evidence of penetration and accumulation of microspheres within extrapulmonary organs. However, in one animal study (23) 1.7% of 3-µm polystyrene particles instilled into the lungs were found in tracheobronchial lymph nodes. In that study, 24 beagle dogs were instilled with 3-, 7-, and 13-µm radiolabeled polystyrene microspheres with a fiberoptic bronchoscope into lung lobes and were followed for 128 d. The biologic half time retention of the 3-µm microspheres was evaluated as 820 d and a few thousand days for 7- and 13-µm microspheres, respectively. The authors concluded that microspheres with a real diameter of 7-µm or more might be retained indefinitely in the lungs of dogs after instillation. In our study, perihilar lymph nodes were examined 10, 20, and 30 d after tracheal instillation of black polystyrene microspheres, and we could not identify the microspheres within the glands, although only two slides from each extrapulmonary organ were examined in nine hamsters. This small number of animals examined does not imply that the same conclusion could be reached with a larger number of hamsters or within other mammals, including humans. Therefore, further safety studies for longer times should be performed before polystyrene microspheres are used for the detection of aspiration in young children. Research using assays for specific food antigens, especially if they can be positive for more than a few days after aspiration, should continue.

Received in original form February 25, 2002

Received in final form June 9, 2002

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