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Home , Choroid plexus papilloma

74 EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008

Case Report Magnetic resonance imaging of a cholesterol granuloma and in a horse B. E. B. MAULET*, M. BESTBIER†, E. JOSE-CUNILLERAS†, J. A. SCRINE‡ AND R. MURRAY† Drève des pinsons 16, B-7850 Enghien, Belgium; †Animal Health Trust, Lanwades Park, Kentford, Newmarket, Suffolk CB8 7UU; and ‡Equine Veterinary Practice, Bognor Road, Horsham, West Sussex RH12 3SH, UK. Keywords: horse; magnetic resonance imaging; cholesterol granuloma; hydrocephalus

Summary duration. On regaining her feet, she had several episodes of ‘petit mal’ (trance-like state), reduced mentation and Magnetic resonance imaging (MRI) was used to a tendency to hold her head to the right. Right panniculus diagnose cholesterol granulomas in the choroid reflexes were reduced and right side weakness was apparent plexuses in the of a horse. This report during the tail-pull test and hopping tests. No significant provides, in addition to the clinical signs and pathology abnormalities were detected using extensive diagnostic findings, a precise description of the MRI appearance of testing; abdominocentesis, rectal palpation, abdominal a cholesterol granuloma. ultrasound, gastroscopy, chest and head radiography. Haematology and biochemistry identified no abnormalities Case history other than mildly elevated inflammatory proteins: serum globulin 41 g/l (rr 26–36 g/l) and fibrinogen 4.0 g/l A 17-year-old hunter-type mare presented with a history of (rr 0.9–3.7g/l). An ELISA test for antibodies to Borrelia intermittent episodes of malaise with depression of 8 months’ burgdorferi was negative. Although lumbosacral cerebrospinal duration. Several short episodes of depression had been noted fluid analysis was blood contaminated, analysis demonstrated by the owner over a longer period but resolved without increased numbers of segmented mature nondegenerate treatment. During the first episode of malaise that required neutrophils once allowance had been made for the blood 9 veterinary intervention, the mare was depressed, pyrexic contamination (red blood cells 0.05 x 10 /l, total number of 9 (40.5°C), tended to stay with the head in a corner (not actually nucleated cell count 0.2 x 10 /l, protein 4.9 g/l, creatine kinase head pressing) and showed one episode of circling to the right. 645 iu/l, glucose 3.9 nmol/l, total number of cells 0.17 x 12 A blood sample taken at the time showed: red blood cells 10 /l). A 4 week course of treatment with doxycycline 3 6.43 x 1012/l (reference range [rr] 5.42–9.62 x 1012/l ), PCV (Doxycycline) 10 mg/kg bwt q. 12 h per os was instigated. 27% (rr 29.2–46.4%), haemoglobin 96 g/l (rr 95–163 g/l), The mare had repeated episodes of depression, approximately every 2–3 days, with lack of awareness of her environment, mean corpuscular volume 51 fl (rr 44–56 fl), mean corpuscular decreased appetite and sometimes appeared centrally visually haemoglobin concentration 306 g/l (rr 308–372 g/l), white impaired with reluctance to lift the right limbs. No abnormal blood cells 7.3 x 109/l (rr 4.8–10.8 x 109/l), platelets 161 x signs had been noted between these bouts. 109/l (rr 113–299 x 109/l). The mare was treated with Because the mare did not improve, she was referred to the dexamethasone (Duphacort Q, 400 mg i.v.)1, oxytetracycline Animal Health Trust for magnetic resonance imaging (MRI) of (Engemycin 10%, 2800 mg i.v.)2 and flunixin meglumine the brain and atlanto-occipital (CSF) (Meflosyl 5%, 600 mg i.v.)1. Major improvement occurred centesis under general anaesthesia. within 2 weeks. During the following 6 months, the mare had one brief Clinical examination episode of depression that improved without treatment within 24 h, but was otherwise normal. After those 6 months On the day of presentation at Animal Health Trust, the mare was (2 months prior to referral), she suffered an episode of colic in good bodily condition but not overweight. She was mildly signs, mild pyrexia and lethargy followed immediately by an lethargic and depressed. A thorough neurological examination episode of syncope with unconsciousness of one hour’s was performed and revealed a slightly decreased menace response on the right side. When the sway test was performed *Author to whom correspondence should be addressed. at the walk on the right, the mare could be pulled off-stride EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008 75

TABLE 1: Pulse sequences parameters used for MR imaging of the brain

Sequences Slice thickness Interslice spacing

3 plane locator 7.0 mm 5.0 mm Sag fast spin echo T2 5.0 mm 2.0 mm Dorsal fast spin echo T2 5.0 mm 2.0 mm Transverse fast spin echo T2 5.0 mm 2.0 mm Transverse gradient echo 3D 4.0 mm 0.5 mm Transverse fast spin echo T2 5.0 mm 2.0 mm Transverse T1 FLAIR 4.0 mm 1.5 mm Transverse T1 FLAIR plus contrast 4.0 mm 1.5 mm T1 plus contrast 5.0 mm 2.0 mm easily. There was intermittent toe dragging in both hindlimbs, Fig 2: Transverse MR image (T2 FSE) showing that the mass in especially when walked with the head up or when walking up the left lateral ventricle caused displacement to the right (R) of the falx cerebri (black arrow) and asymmetric compression of and down hill, and the mare was very bouncy in the transitions the pons region (white arrow). L = left; R = right. from trot to walk; all these signs indicated mild ataxia.

MRI results

MR imaging of the brain was performed using a high-field 1.5 T GE Signa Echospeed magnet4. The horse was placed in right lateral recumbency under general anaesthesia, with the head positioned as centrally as possible within an integrated quadrature body radiofrequency coil. Sagittal, dorsal and transverse plane MR images were obtained using 2D T2 weighted fast-spin echo (FSE), 3D T2* gradient echo (GRE) and 2D transverse T1 fluid attenuated inversion recovery (FLAIR) sequences obtained pre- and post contrast administration (20 ml of gadobenate dimeglumine i.v., MultiHance, 1 ml contains gadobenic acid 334 mg [0.5 mol/l] as dimeglumine salt [gadobenate dimeglumine 528 mg = gadobenic acid {334 mg} + meglumine {195 mg}])5 (Table 1). Fig 3: Dorsal MR image (T2 FSE) demonstrating asymmetric Sagittal images of the brain at the level of the third widening of the lateral ventricles (most pronounced in their ventricle showed an ovoid well-circumscribed mass centred in aboral aspect) due to obstructive hydrocephalus. There is the left lateral cerebral ventricle (Fig 1), and a smaller round compression of the brain parenchyma and cerebellum (arrow). mass in the right lateral cerebral ventricle. The mass in the left There was a smaller mass in the right ventricle. L = left, R = right. lateral ventricle was causing displacement to the right of the

Fig 4: Transverse MR image (T2 GRE) illustrating that the mass had mostly void signal on this sequence. A focus of increased signal was seen in the left side mass (arrow), which was Fig 1: Sagittal MR image (T2 FSE) of the brain showing an ovoid interpreted as the presence of small pockets of fluid and/or well-circumscribed mass centred in the left lateral cerebral disorganised soft tissue within the stroma of this mass. There is ventricle. a smaller mass in the right ventricle. L = left, R = right. 76 EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008

Fig 5: Transverse MR image (T1 FLAIR): the mass is slightly hypointense relative to other parenchyma, which was interpreted as presence of magnetic susceptibility artefact and likely presence of haemosiderin within the mass. The presence of haemosiderin was confirmed post mortem. L = left, R = right. Fig 7: Section through the cerebrum showing the left lateral ventricle intraventricular mass.

Areas of increased signal intensity within the left subcortical parenchyma were observed on T2 weighted images, which was interpreted as increased fluid accumulation or oedema of the neural tissue compressed by this mass (Fig 3). The smaller mass in the right ventricle measured 2.0 x 1.5 x 1.0 cm (Figs 3 and 4). There was evidence of slight signal enhancement in the mass following contrast administration (Fig 6). An irregular/granular structure of the mass was apparent in all images (Figs 1–6) and particularly in the images post contrast administration (Fig 6). A CSF sample was obtained from the atlanto- occipital cistern under general anaesthesia and had demonstrated marked elevation in total protein (2.5 g/l, Fig 6: Post contrast transverse MR image (T1) revealing slight 6 signal enhancement in the mass following contrast rr 0–1.2 g/l). The nucleated cell count was 3 x 10 /l, and administration. L = left, R = right. consisted of nondegenerate neutrophils, lymphocytes and occasional monocytes. falx cerebri and asymmetric compression of the pons region Following the procedure, the horse had an uneventful (Fig 2), cerebellum and brain parenchyma (Fig 3). There was recovery from anaesthesia. In view of the size and location of asymmetric widening of the lateral ventricles (most the mass, the prognosis was considered hopeless, so after pronounced in their caudal aspect) due to obstructive discussion with the owner, the horse was subjected to hydrocephalus (Fig 3). The mass in the left lateral ventricle euthanasia. measured 5.8 x 4.0 x 3.3 cm. Most of the mass had void signal on gradient echo images (Fig 4), low signal on T2 weighted images (Figs 1–3), and it was isointense to slightly hypointense relative to other parenchyma (white matter) on T1 spin echo and FLAIR images (Fig 5), which was interpreted as presence of a magnetic susceptibility artefact and likely presence of haemosiderin within the mass. In addition, this mass appeared heterogeneous in its signal intensity and a focus (approximately 0.5 x 0.5 cm) of increased signal intensity was seen predominantly in T2 weighted images (Fig 3) and to a lesser degree in gradient echo scans (Fig 4), which probably represented the presence of small pockets of fluid and/or disorganised soft tissue within the stroma of this mass. The mass was surrounded by cerebrospinal fluid in all 3 planes, suggesting lack of adhesion formation or infiltration of the mass to Fig 8: Transverse section through the cerebrum: closer view of surrounding subcortical parenchyma (Figs 1–3). bilateral intraventricular mass (cholesterol granuloma). EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008 77

namely an inflammatory granulomatous process affecting the of especially the lateral ventricles, with marked infiltration and deposition of cholesterol within the tissues (Jubb and Huxtable 1993). The choroid plexus has an epithelial cuboidal cell lining and the occurs within the choroid plexus membranes, resulting in expansion of the membranes and filling of the space. Therefore, an outer layer of epithelial cells is all that remains of the choroid plexus. Cholesterol granulomas have been described in the horse in several reports (Ooms et al. 1975; Ivoghli et al. 1977; Carnero et al. 1979; Rooney 1979; Johnson et al. 1993; Duff 1994; Jackson et al. 1994; King 1997; Hahn et al. 1999; Laugier et al. 2004); however, MRI appearance has not. Amongst domestic animals, tumours of the choroid plexus including papilloma and carcinoma, are rare and have an incidence of approximately 9% of primary central nervous Fig 9: Lymphocytic and plasma cell infiltration with haemosiderophages, cholesterol clefts and fibrous connective system (CNS) tumours (Koestner and Higgins 2002). Pirie et al. tissue stroma. H/E section, 10x magnification. (1998) reported one case of choroid plexus papilloma in a 5-year-old Warmblood mare with neurological symptoms. Cholesterol granulomas (or cholesteatosis) of the choroid Pathological findings plexuses are tumour-like nodules. Reported prevalence varies, and is up to 15–20% of old horses (Jubb and Huxtable 1993). Removal of the brain and external examination did not reveal In a study of 1771 horses that underwent necropsy, only any significant gross abnormalities. On incision into the left and 1.86% had cholesterol granuloma (representing 16.42% of right ventricles a moderate amount of slightly yellow-tinged the total of the tumours), with a slightly higher frequency in fluid was released. Opening of the ventricles revealed large horses older than 10 years compared with younger horses bilateral, smooth, rounded brown intraventricular masses with (Laugier et al. 2004). This places cholesterol granulomas in the fine pale stippling of their surfaces (Fig 7). The brain was fixed position of third most commonly found neoplastic or in formalin and subsequently multiple transverse incisions were neoplastic-like lesion in horses, after the thyroid adenoma and made to reveal an oval mass within the left lateral ventricle lipoma, but before the lymphomas, sarcoids and melanomas extending across and into the right ventricle (Fig 8). The left (Laugier et al. 2004). and right ventricles were moderately distended (hydrocephalus) Most reported cholesterol granulomas are small and do with the left ventricle extending further caudally than normal not cause any clinical signs (Rooney 1979; Johnson et al. and abutting into the cerebellum. Cut surfaces of the masses 1993; Hahn et al. 1999; Laugier et al. 2004). However, if the were brown, with multifocal pale white-yellow speckling and a cholesterol granuloma reaches a significant size, reported fine gritty texture (Fig 8). clinical signs associated with it are very variable and Histological examination of the mass revealed an intermittent, as was the case here, with periods of remissions expansile, encapsulated mass composed of multiple angular between the symptomatic episodes. The discontinuous aspect clefts and large open rounded areas (cholesterol clefts and of the signs is commonly reported in the literature (Ooms et al. fatty deposits) within a moderate fibrous connective tissue 1975; Johnson et al. 1993; Duff 1994; Jackson et al. 1994; network (Fig 9). There was a large number of thin-walled Vink-Nooteboom et al. 1998; Hahn et al. 1999). This could be blood vessels within the fibrous connective tissue stroma, with due to intermittent hydrocephalus and/or oedema formation expansion of tissues by a clear slightly eosinophilic fine within the cerebral parenchyma, with remission taking place granular material (proteinaceous oedema). There was a mild to once the pressure of the CSF returns to normal. However, moderate diffuse lymphocytic and plasma cell infiltration, with Johnson et al. (1993) suggest that it could be due to moderate numbers of haemosiderin-containing macrophages intermittent release of irritant substances from the cholesterol scattered throughout the tissue (Fig 9). There were multifocal granulomas. Depression is a clinical sign that has been accumulations of aged neutrophils and multifocal areas of mild frequently reported (Ooms et al. 1975; Johnson et al. 1993; intrastromal haemorrhage. These findings were consistent with Jackson et al. 1994; King 1997; Vink-Nooteboom et al. 1998; a diagnosis of cholesterol granuloma. Hahn et al. 1999). Other signs observed and that have been described in the Discussion literature are behaviour changes (Ooms et al. 1975; Johnson et al. 1993; Duff 1994; Hahn et al. 1999; Laugier et al. 2004), Cholesterol granulomas are tumour-like masses of the choroid syncope-like attacks (Ooms et al. 1975; Vink-Nooteboom et al. plexuses of the ventricles of the equine brain. Cholesterol 1998; Hahn et al. 1999), apparent intermittent blindness granuloma, cholesteatoma and cholesteatosis of the choroid (Jackson et al. 1994), head turn (Ooms et al. 1975; Rooney plexus are different names referring to the same process, 1979; King 1997), lack of awareness to the environment 78 EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008

(Johnson et al. 1993; Duff 1994; Jackson et al. 1994), circling 1993). Hahn et al. (1999) hypothesised that cholesterol (Johnson et al. 1993; Jackson et al. 1994; Vink-Nooteboom granuloma might represent a chronic granulomatous reaction et al. 1998), ataxia (Ivoghli et al. 1977; King 1997; Vink- to cholesterol crystal deposition, associated with chronic Nooteboom et al. 1998; Hahn et al. 1999) and vague signs of choroid plexus vascular leakage. They are more frequent in the colic (Johnson et al. 1993; Vink-Nooteboom et al. 1998). plexus from the than in the lateral ventricle, Other symptoms described in horses with cholesterol although those in the lateral ventricle are more important as granulomas, which were not observed in the case presented they can attain a large size (Jubb and Huxtable 1993; Hahn here, include a tendency to aggressiveness and intractability et al. 1999), and will potentially obstruct the foramina of (Rooney 1979), weakness (Hahn et al. 1999), motor paralysis Monro, causing hydrocephalus and secondary atrophy of the (Ivoghli et al. 1977), unresponsiveness to visual and tactile peripheral (Ooms et al. 1975; Johnson et al. stimulations (Johnson et al. 1993; Duff 1994), head tilt, 1993; Laugier et al. 2004), as was the case here. A refusing to turn towards one side (Ooms et al. 1975; Rooney symptomatic cholesterol granuloma present in the third 1979; Jackson et al. 1994; King 1997), yawning (Johnson ventricle has been reported (Rooney 1979). et al. 1993), head pressing (Johnson et al. 1993), generalised The gross pathological appearance of the cholesterol seizures (Ivoghli et al. 1977; Jackson et al. 1994; Hahn et al. granuloma described here corresponded to the descriptions in 1999), bilateral dorsal strabismus (Johnson et al. 1993), bizarre the literature as well-circumscribed, firm, granular and posture (Johnson et al. 1993; Jackson et al. 1994), head yellowish-brown (Ooms et al. 1975; Ivoghli et al. 1977; Hahn (Jackson et al. 1994), low head carriage (Ooms et al. et al. 1999; Laugier et al. 2004). Histology clearly showed the 1975; Jackson et al. 1994) or high head carriage in presence of multiple angular clefts and large open rounded combination with upwards movements (Vink-Nooteboom areas containing cholesterol crystals within a fibrous et al. 1998), facial nerve paralysis (Vink-Nooteboom et al. connective tissue network. This corresponds to the description 1998), anisocoria (Vink-Nooteboom et al. 1998), reluctance to generally found in other reported cases (Ooms et al. 1975; move (Ooms et al. 1975; Jackson et al. 1994; Hahn et al. Vink-Nooteboom et al. 1998; Laugier et al. 2004). A 1999), stiffness at the walk (Jackson et al. 1994), drifting to granulomatous reaction and haemosiderin pigments were one side at the walk (Ooms et al. 1975), anorexia observed in the macrophages; these features have also been (Vink-Nooteboom et al. 1998), sweating (Johnson et al. 1993; reported (Ooms et al. 1975; Vink-Nooteboom et al. 1998). Vink-Nooteboom et al. 1998), muscle fasciculations (Johnson Some oedema was present and there was a granulomatous et al. 1993). The majority of those signs indicate forebrain inflammation with cholesterol clefts. This consisted of a mild pathology. Jackson et al. (1994) reported 3 cases where all to moderate diffuse lymphocytic and plasma cell infiltration 3 horses were obese; however, this was not the case either with moderate numbers of haemosiderin-containing here or in most other case reports. macrophages scattered throughout the tissue. There were The CSF sample obtained just after the MRI examination multifocal accumulations of aged neutrophils and multifocal demonstrated marked elevation in total protein (2.5 g/l, areas of mild intrastromal haemorrhage. rr 0–1.2 g/l) and elevation of neutrophils. Jackson et al. (1994) Nowadays MRI is increasingly used as a diagnostic tool for also reported raised protein concentration in CSF samples orthopaedic conditions, but its use for diagnostic imaging in taken from 2 horses with cholesterol granuloma. The neurological conditions in horses has also been reported markedly elevated protein concentration and the presence of (Ferrell et al. 2002; Spoormakers et al. 2003; Audigie et al. small numbers of neutrophils found in the horse described in 2004) and has already proven useful in diagnosing brain this report suggested the presence of an inflammatory process masses (Spoormakers et al. 2003; Audigie et al. 2004). MRI of affecting the , with possible blockage of CSF flow. the normal brain has been described (Arencibia et al. 2001; Neither neoplastic cells nor infective agents were recognised in Vazquez et al. 2001). One of the main advantages of MRI the horse described in this report; however, inflammation compared with other imaging modalities is the excellent soft secondary to neoplasia was not excluded, as only a minority of tissue discrimination of the images (Westbrook and Kaut CNS tumours will exfoliate into the CSF. Ooms et al. (1975) 1998). MRI appearance of cholesterol granulomas has not reported that the CSF sample of the horse described in their been previously described. The cholesterol granuloma was report had a normal protein and nucleated cell count; well defined on all the sequences used (Table 1 and Figs however, it was mildly yellow-tinged and they hypothesised 1–6). The use of different sequences helps in characterising that this was due to the presence of haemosiderin. The the structure of the mass. The mass predominantly had void cerebrospinal fluid that escaped from the ventricles of our signal on GRE images, low signal intensity on T2 weighted specimen at post mortem examination was also yellow-tinged images and was hypointense on T1 spin echo and FLAIR and we hypothesised that this was associated with high images (Figs 1–5), which suggested the presence of protein levels (markedly elevated in the premortem CSF haemosiderin within the mass. The irregular/granular sample: 2.5 g/l) although the presence of haemosiderin might structure of the mass was apparent in all images but also have contributed to the colour. particularly in the images after contrast administration The development of cholesterol granulomas could be (Fig 6). MR images were obtained in the sagittal, dorsal and related to chronic or intermittent oedema and congestive transverse planes in order to allow a precise definition of the haemorrhages in the choroid plexuses (Jubb and Huxtable shape and position of the mass (Figs 1–6). EQUINE VETERINARY EDUCATION / AE / FEBRUAry 2008 79

The scanning sequences used to image the brain of this Ferrell, E.A., Gavin, P.R., Tucker, R.L., Sellon, D.C. and Hines, M.T. case are shown in Table 1. As a contrast agent, we used (2002) Magnetic resonance for evaluation of neurologic disease in gadolinium, which is a rare earth heavy metal (Westbrook and 12 horses. Vet. Radiol. Ultrasound 43, 510-516. Kaut 1998) and is the most commonly used contrast agent Hahn, C.N., Mayhew, I.G.(Joe) and Mackay, R.J. (1999) Diseases of the (McRobbie et al. 2003). It enhances the signal in the tissues in forebrain. In: Equine Medicine and Surgery, 5th edn., Eds: P.T. Colahan, A.M. Merritt, J.N. Moore and I.G. (Joe) Mayhew, Mosby, which it accumulates, and therefore highly vascular tumours, St Louis. pp 904-913. for example, will become brighter (McRobbie et al. 2003). In addition, if the blood-brain barrier is disrupted, gadolinium Ivoghli, B., Emady, M. and Rezakhani, A. (1977) Motor paralysis associated with cholesteatoma in a mare. Vet. Med. small anim. will leak into the lesion and enhance the area (Westbrook and Clin. 72, 602-604. Kaut 1998; McRobbie et al. 2003). Common clinical Jackson, C.A., Delahunta, A., Dykes, N.L. and Divers T.J. (1994) indications for contrast agents include tumours, infection, Neurological manifestation of cholesterinic granulomas in three infarction, inflammation and post traumatic lesions horses. Vet. Rec. 135, 228-230. (Westbrook and Kaut 1998). Post contrast injection, there was Johnson, P.J., Tsang, L.L. and Jennings, D.P. (1993) Diffuse cerebral evidence of slight increase in signal intensity in the masses. associated with hydrocephalus and cholesterinic This was to be expected as the choroid plexuses are one of granulomas in a horse. J. Am. vet. med. Ass. 203, 694-697. the extra-axial areas outside the blood-brain barrier that Jubb, K.V.F. and Huxtable, C.R. (1993) The . In: shows normal enhancement after contrast administration Pathology of Domestic Animal, Volume 1, 4th edn., Eds: K.V.F. (Westbrook and Kaut 1998). Jubb, P.C. Kennedy and N. Palmer, Academic Press, Inc. San Diego. MRI demonstrated a well-defined space occupying mass pp 267-440. within the left lateral cerebral ventricle and an associated King, J.M. (1997) Bilateral choroid plexus cholesteatomas. Vet. Med. smaller mass in the right lateral ventricle, which caused 92, 514. shifting of the cerebral parenchyma, compression of the pons Koestner, A. and Higgins, R.J. (2002) Tumours of the nervous system. region and cerebellum as well as obstructive hydrocephalus. In: Tumours in Domestic Animals, 4th edn., Ed: D.J. Meuten, Iowa Due to MR signal density and the localisation of the masses, State Press, Blackwell, Iowa. pp 697-738. there was a strong suspicion that it would be a cholesterol Laugier, C., Tapprest, J., Foucher, N., Doux, N., George, C., Longeart, granuloma, although abscesses or a neoplastic masses could L. and Net, J.L. (2004) Prevalence of equine tumours in 1771 not be completely ruled out. Abscesses were considered horses examined post-mortem. Prevalence de la pathologie unlikely on the basis of the CSF sample results. tumorale dans un effectif de 1771 chevaux autopsies. Prat. vét. Equine 36, 21-35. Diagnosis of a cholesterol granuloma in a horse is a challenge, especially in view of the intermittent and very McRobbie, D.W., Moore, E.A., Graves, M.J. and Prince, M.R. (Eds) (2003) Seeing is believing: introduction to image contrast. In: MRI variable signs presented, with nonspecific haematology and From Picture to Proton, Cambridge University Press, Cambridge. CSF sample results. 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