Quantitative Fecal Indium Ill-Labeled Leukocyte Excretion in the Assessment of Disease in Crohn's Disease

Transcription

1 GASTROENTEROLOGY 1983;85: Qantitative Fecal Indim Ill-Labeled Lekocyte Excretion in the Assessment of Disease in Crohn's Disease S. H. SAVERYMUTTU, A. M. PETERS, J. P. LAVENDER, M. B. PEPYS, H. J. F. HODGSON, and V. S. CHADWICK Department of Medicine and Diagnostic Radiology. Royal Postgradate Medical School, Hammersmith Hospital, London W12, United Kingdom The assessment of disease activity in Crahn's disease involves determination of either clinical indexes (e.g., Crahn's disease activity index) or laboratory measrements (e.g., C-reactive pratein and erythracyte sedimentation rate). These have the disadvantage of being indirect and nonspecific correlates of gt inflammation. We have assessed disease activity in Crahn's disease by measrement of fecal lekocyte excretion after intravenos administration of either 111 In-labeled mixed lekocyte or pre granlocyte preparations. With mixed lekocyte preparations, fecal excretion of radioactivity correlated with Crahn's disease activity index (r =.78, P <.1) and C-reactive protein (r =.74, P <.1). Using pre granlocytes, fecal 111 In excretion (range 1.5%-52%) was mch higher than with mixed lekocytes (range.1 %-11.%), showing significant correlations with Crahn's disease activity index (r =.731, P <.1), C-reactive pratein (r =.716, P <.1), and erythracyte sedimentation rate (r =.676, P <.1). Qantitative fecal excretion of 111 In-lekocytes is a new method of assessing disease activity in Crahn's disease, specific for bowel inflammation and sitable for objective assessment of disease activity in therapetic trials. The assessment of disease activity in Crohn's disease is notoriosly difficlt. A change in clinical state may be de to one or more of a nmber of factors, inclding inflammation within the gt wall, sperimposed infection, fibros strictres, and fistla Received Jly 16, Accepted Jne 1, Address reqests for reprints to: Dr. S. H. Saverymtt, Department of Medicine, Royal Postgradate Medical School, Dcane Road, London W12 OHS, United Kingdom. This stdy was fnded in part by the Wellcome Trst (Dr. Saverymtt) and the Cancer Research Campaign (Dr. Peters) by the American Gastroenterological Association /83/$3. formation. Varios clinical indexes have been developed that assess the severity of disease by evalating a nmber of clinical parameters. The Crohn's disease activity index (CD AI) (1) is the most widely sed of the clinical indexes and proved sefl in the National Cooperative Crohn's Disease Stdy by allowing assessment of clinical severity in patients from a wide variety of participating instittions. The index has been criticized becase of its dependence on sbjective vales and also becase it is inflenced by noninflammatory factors. Becase medical therapy in Crohn's disease is directed primarily against the inflammatory component, there have been several approaches in assessing this factor. In an effort to avoid sbjective bias, several grops have investigated laboratory tests that reflect inflammatory activity, and erythrocyte sedimentation rate (ESR) (2), serm orosomcoid (3), and C-reactive protein (CRP) (4) have been shown to correlate with disease activity in Crahn's disease. Frthermore, measrements of gt protein loss (5) are sefl and apparently correlate well with extent and severity of disease. However, none of these objective measrements is specific for bowel inflammation. An objective and specific assessment of the inflammatory component in disease activity cold be provided by a detailed histologic srvey of mltiple mcosal biopsy specimens; however, becase Crohn's disease may affect any part of the bowel (6,7) this approach wold be impractical and nsitable for repeated measrements. An alternative approach is to qantitate the lekocyte efflx from inflamed bowel. Recently, preliminary reports (8,9), sing a mixed lekocyte preparation labeled with 111 In, have shown that these cells localize rapidly in inflamed bowel, emigrate to the bowel lmen, and are excreted in feces, providing the basis for fecal qantitation. In this stdy, we have prospectively compared

2 1334 SAVERYMUTTU ET AL. GASTROENTEROLOGY Vol. 85, No. 6 qantitative fecal 111 In-lekocyte excretion in Crohn's disease with clinical and laboratory assessments of disease activity sing both mixed lekocyte preparations and, for the first time, pre granlocyte preparations. Patients and Methods Patients Fifty-two stdies were performed in 28 patients with Crohn's disease. The diagnoses were based on standard clinical, radiologic, and histopathologic featres, the last according to the criteria of Lockhart-Mmmery and Morson (1,11). Twenty-five patients with diarrhea de to irritable bowel syndrome were also stdied. All patients gave written informed consent, and the stdy was approved by the hospital ethics committee. Clinical and Laboratory Assessment Stdies of fecal ll1in-lekocyte excretion in patients with Crohn's disease were performed immediately after determination of the CDAI. Patients were assigned to an active (CDAI > 15) or inactive (CDAI < 15) grop. An estimation of ESR (Westergren) (12) and serm CRP (13) was made at the time of each fecal lekocyte excretion measrement. Simltaneos fecal l1lin-lekocyte stdies and measrements of gt protein loss sing the SlCr techniqe (14) were performed in 11 patients with Crohn's disease. Stdies of Labeled Lekocyte and Protein Excretion Cell separation and labeling. All procedres were carried ot aseptically, sing sterile containers and soltions, in a laminar flow cabinet (Microflow-Pathfinder, model 2229). Two cell preparations were sed, mixed lekocytes (31 stdies) and pre granlocytes (46 stdies). "MIXED" LEUKOCYTES. Sixty milliliters of venos blood was drawn into two sterile, disposable plastic syringes containing 5.5 ml of acid citrate dextrose (ACD) (National Instittes of Health Formla A) each and dispensed into 25-ml polystyrene tbes (Sterilin). Hydroxyethyl starch in.9% sodim chloride (Fresenis) was added to give a final concentration of.6% vol/vol and the blood was incbated at 37 C for 6 min. The spernatant containing the lekocytes was centrifged at 1 g for 5 min to yield a "mixed" cell pellet. The cell pellet was resspended in 8 ml of.15 M NaCl containing dextrose (1 part 5% dextrose to 1 parts NaCl), ACD (1 part to 1 parts NaCl), acetylacetone (.19%), and 2 mm of HEPES bffer (ph 7.6). Approximately 2 p,ci of lllincl in.4 M HCl (The Radiochemical Centre, Amersham, UK.) in <.25 ml was added, and the mixtre was left nder gentle agitation on a roller for 1 min. Five milliliters of cell-free plasma was then added, and the cells were sedimented by centrifgation at 1 g for 3 min. The spernatant was discarded and the cells resspended in 5 ml of plasma for reinjection. The labeling efficiency averaged >8%. In six stdies, the range of ratios of erythrocytes to granlocytes in the labeled cell preparation was :1 (1.7 ±.8:1, mean ::': SD). The mean range (±SD) of activity on the individal cell fractions was, for granlocytes, 22%-39% (31.5 ± 6.3); for erythrocytes, 9.5%-49% (29.7 ± 13.9); for lymphocytes and platelets, 24%-45% (34.1 ± 1.2); and for plasma,.9%-7.% (4.5 ± 2.2). The mean nmber of lekocytes was 21.2 x 1 7 (range x 1 7 ). "PURE" GRANULOCYTES. Mixed lekocytes were isolated from 6 ml of whole blood as described previosly. The granlocytes were then isolated by sedimentation on discontinos density-gradient colmns made p of mixtres of atologos plasma and either (a) Percoll (11 stdies) or (b) metrizamide (35 stdies). Fll details of the Percolllplasma separation are described elsewhere (15). Briefly, three densities were prepared by dilting isoosmolar Percoll (9 volmes of Percoll to 1 volme of 1.5 NaCl, specific gravity 1.12 g/ml) with cell-free plasma to give 65%, 6%, and 5% vol/vol (Percolllplasma) soltions. Two milliliters of each soltion was then careflly layered into 1-ml Sterilin polystyrene test tbes (p to for tbes, depending on the size of the mixed-cell pellet) and "mixed" white cells in 2 ml of plasma were layered on top of each tbe. The gradients were spn at 2 g for 5 min and the majority of the granlocytes were recovered from the interface between the 6% and 65% bands. For metrizamide/plasma, a stock of 35% wt/vol metrizamide in water, specific gravity g/ml (at 2 C), was dilted with plasma to give 5% and 4% vol/vol metrizamide/ plasma soltions, respectively. Two milliliters of each soltion was layered into 1-ml Sterilin polystyrene test tbes, followed by 2 ml per tbe of the "mixed" white cells, and then centrifged at 2 g for 5 min. The granlocytes were recovered from the interface between the two densities. The granlocytes isolated by both procedres were washed once with excess plasma and resspended in.5-1 ml of cell-free plasma. Tropolone (Flka), at a concentration of 4.4 x 1~3 M in HEPES saline bffer (ph 7.6) containing 2 mm of HEPES in.8% vol/vol sodim chloride in a volme of 1 p,l, was added, followed by ~3 p,ci of l1lincl in <5 p,l.4 M HCl. The final plasma concentration was not <8%. After 5 mintes' incbation at room temperatre, 5-1 ml of cellfree plasma was added, and the cells were centrifged at 1 g for 5 min. After resspensioll in frther plasma, the cells were ready for reinjection. The labeling efficiency was 5%-8%, and the injected dose averaged 2 p,ci. The total body radiation was.4 rad, and the radiation dose to the target organ-the spleen (16)-was estimated to be between 1 and 3 rads (16,17). "Pre" granlocytes isolated by both methods contained 2%-2% erythrocytes bt no platelets or mononclear cells. Becase of the mch higher affinity of l1lin-tropolonate for granlocytes than erythrocytes (Danpre HI. Osman S, npblished observations), the fraction of the dose present on erythrocytes was estimated to be <3%. Less than 8% of ll1in activity on the labeled cell preparation was plasma associated. The remaining activity was firmly bond; after repeated washing in plasma for p to

3 December 1983 FECAL l11in-leukocyte IN CROHN'S DISEASE 1335 Table 1. Disease Distribtion in Patients With Crohn's Disease Site of disease Region of bowel resected Nmber of patients Pancolitis 5 Transverse colon and 2 descending colon Ileocolonic 5 Small bowel only 8 Ileostomy stmp Colon 1 Ileocolonic anastomosis Terminal ilem 4 Terminal ilem, as- 3 cending and transverse colon three times, there was <1% eltion. After leaving the labeled granlocytes in plasma for 4 h, there was <1% eltion. The range of granlocytes injected was 4-1 x 1 6 cells. In vitro tests of granlocyte fnction, chemotaxis, and phagocytosis showed no difference between nlabeled and labeled granlocytes. Protein losing stdies. Approximately 1 p,ci of 5lCrC13 (The Radiochemical Centre, Amersham) was injected in 1 ml of isotonic saline (14) at the same time as the 11lIn-labeled mixed lekocytes. The total body radiation dose was ~.7 rad (18). Estimation of fecal radioactivity. After administration of labeled cells, a 4-day fecal collection was made in daily aliqots. In those patients receiving labeled cells only, the total 111In content of each daily aliqot was conted on an ARMAC gamma conter and expressed as a percentage of the injected dose, after a decay correction. The distribtion of 11lIn activity between particlate and solte fractions of the feces was determined by mixing ~.5 g of feces in 1 ml of.9 M NaCl and estimating the activity in the sspension and then that of the spernatant obtained after centrifgation at 2 g for 1 min. In those stdies where simltaneos protein and lekocyte excretion was performed, a correction for cross-reactivity was made. The daily aliqots were conted at the end of the 4- day collection and again after an interval of 35 days, at which time the 111In activity (half-life 67 h) had decayed to.1% of initial activity. Chromim 51 activity (half-life 28 days) was corrected to initial vales and initial IllIn activity was calclated. Granlocyte kinetics. Blood samples were ob- tained at intervals p to 48 h after injection of the labeled cells in 15 patients receiving pre granlocytes. The cellbond and cell-free IllIn was determined in each sample. Recovery vales and granlocyte half-life estimations were calclated (19). Granlocyte absorption. The absorption of orally administered granlocytes was investigated in three stdies: a patient with diarrhea de to jejnal diverticlosis, a patient with metastatic liver infiltration withot diarrhea, and a normal volnteer sing total body conting. Statistics Statistical analysis was performed sing the Wilcoxon rank sm test and Spearman's rank correlation coefficient. All p vales >.5 were considered not significant. Reslts Patient Details The distribtion of disease in the patients with Crohn's disease is shown in Table 1. Patients with overt sepsis, sch as perianal abscess or infected fistla, were exclded from the stdy. Mean vales and range for CDAI, CRP, and ESR for the active or inactive grops receiving either mixed lekocyte or pre granlocyte preparations are shown in Table 2. In 2 stdies, patients were receiving therapy with prednisolone, azathioprine, slphasalazine, or antibiotics at the time of the stdy. "Mixed" lekocyte stdies and comparison with labeled protein excretion. The normal range for fecal lekocyte excretion for patients receiving mixed lekocyte preparations was established in 11 patients with the irritable bowel syndrome. Mean 4- day excretion of l11ln was.46% ±.3 (mean ± SD). In 2 stdies in patients with Crohn's disease receiving mixed lekocyte preparations, fecal excretion ranged from.2% to 11 % with excretion significantly higher in the active grop compared with the inactive grop (p <.1) and patients with the irritable bowel syndrome (p <.1) (Figre 1). The Table 2. Clinical Details and Cell Preparation Used Cell Diseases of patients CD AI preparation Crohn's disease <15 Mixed Pre >15 Mixed Pre Irritable bowel syndrome Mixed Pre n ESR in first CDAI" CRP (f.lg/ml)" hor a 119 ± ± ± 7 11 ± ± ± ± ± 38 4 ± ± ± ± ± ± 2.8 CDAI = Crohn's disease activity index. CRP = C-reactive protein. ESR = erythrocyte sedimentation rate. a Mean ± SD.

4 1336 SAVERYMUTTU ET AL. GASTROENTEROLOGY Vol. 85, No. 6-52, - c: '" x '" ro.. '" I * ~ ~, I 8 Active Inactive I B S C ro h n s C ro h n s Figre 1. Fecal 111In excretion in active Crohn's disease, inactive Crohn's disease, and the irritable bowel syndrome (IBS) for patients receiving mixed lekocytes (open symbols) or granlocytes (closed symbols). difference in excretion between the irritable bowel syndrome grop and the inactive grop was not significant (.1 > p >.5). There were significant correlations between fecal lekocyte excretion and CD AI (r =.78, P <.1) (Figre 2) and CRP (r =.72, P <.1). The correlation with ESR (r =.396, P >.5) failed to reach significance. In 11 stdies, gastrointestinal protein loss was measred simltaneosly with fecal lekocyte excretion and was significantly correlated (r =.74, P <.2) (Figre 3). A comparison of the kinetics of lekocyte and protein excretion showed a greater percentage excretion and more rapid fecal excretion of cells than of protein (Figre 4). "Pre" granlocyte stdies: Granlocyte ki- netics. Mean (± SD) recovery of granlocytes at 3-4 min after reinjection of the cells was 32.8% ± 7.7 (21%-46%). Mean (± SD) half-life was 6.8 h ± 2.1 h ( h). Mean (± SD) plasma activity was 2.6% ± 1.3 at 3-4 min and did not rise over the sbseqent 48 h. After oral administration of lllin_ granlocytes in three stdies, there was.3%,.25%, and.8% whole body retention of initial activity after 4 days. There was no detectable activity in blood or 4-day rine collections. The normal range for fecal lekocyte excretion in the 14 patients with the irritable bowel syndrome receiving pre granlocyte preparations was (.85% ±.4, mean ± SD). In 32 stdies sing pre granlocyte preparations, fecal lekocyte excretion was higher than with the mixed lekocyte preparations, ranging from 1.5% to 52%. Eighty percent :±: 5% (mean ± SD) of the fecal 111 In activity was in the particlate phase. Fecal excretion was higher in the active grop compared with the inactive grop (p <.1) and with patients with the irritable bowel syndrome (p <.1) (Figre 1). There was only 1 patient in the active grop with a fecal lekocyte excretion that overlapped with the irritable bowel syndrome grop. Fecal excretion was significantly higher in the inactive grop than in the irritable bowel syndrome grop (p <.1). Correlations between fecal excretion and CRP (r =.716, P <.1), CDAI (r =.731, P <.1) (Figre 2), and ESR (r =.676, P <.1) were all significant. In the majority of individal cases, fecal lekocyte excretion confirmed the disease activity indicated by CRP and ESR measrements. In 2 patients, there was a raised fecal lekocyte excretion in the presence of a normal erp ( /Lg/ml) and a normal ESR. Both patients were clinically active (CDAI > 15) on treatment and, in 1 patient with colonic disease, histology was available and showed c '" x '" -~ 36 ~ '" ~ if(.". 15 COAl 3 45 Figre 2. Fecal 111In vs. Crohn's disease activity index (CDAI) in patients receiving mixed lekocytes (open symbols) or granlocytes (closed symbols). 12 g r '" ~ ~ ~4 ~. ()-<' I 2 4 %F ecal Drotein excretion r O. 74 p<o.1l2 Figre 3. Fecal 111 In-lekocyte excretion vs. fecal 51Cr-protein excretion in patients receiving mixed lekocytes and simltaneos OlCrC13. I 6

5 December 1983 FECAL ll1in-leukocyte IN CROHN'S DISEASE 1337 ' ~ 2 o * o Days Figre 4. Kinetics of 111 In-lekocytes (closed circles) and 51Cr_ protein (open circles)' excretion. Crves represent cmlative excretion based on percentage of 4-day total radioactivity excreted per day. Asterisk represents a significant difference between lekocyte and protein excretion (p <.5). active colitis. In serial stdies sing 111 In-granlocyte preparations in 8 patients with active disease who improved on treatment, there was a mean decrease of 41.8% in CD AI associated with a mean 57.6% decrease in fecal granlocyte loss. Individal stdies showed a parallel decrease in CDAI and fecal granlocyte loss (Figre 5). Discssion Qantitative fecal 111 In-lekocyte excretion measrement is a new method of assessing inflammatory activity in Crohn's disease and shows a significant correlation with established clinical and laboratory indexes of disease activity. Serial stdies on individal patients sggest that this method can monitor therapy. It is safe in severely ill patients who tolerate intensive investigation badly. In addition to providing an assessment of disease activity based on the fecal excretion, imaging over the abdomen after injection of labeled cells permits assessment of regional bowel involvement and detects any complicating abscesses (8,9). The ideal measrement for the assessment of disease activity in Crohn's disease shold be easy to perform, specific for inflammatory bowel disea5e, and shold respond qickly to changes in activity. Clinical indexes of activity may be heavily infl~ enced by sbjective factors, sch as the effect of srgical resections or fibros strictres. Althogh laboratory indexes, sch as ESR and the acte phase reactants CRP and orosomcoid, may correlate with Crohn's disease activity, none is specific for bowel inflammation and so may be inflenced by associated disease, extraintestinal complications, and intercrrent illness. Protein loss into the bowel correlates with CDAI (5). The measrement is most accrately qantitated as a clearance, bt this has the disadvantage of reqiring 8-11 days to complete (2). Frthermore, bowel protein loss is not specific for bowel inflammation, as it may be inflenced by noninflammatory factors sch as secondary intestinal lymphangiectasia and bacterial overgrowth. Despite these limitations, it is crrently regarded by many as the best assessment of disease activity in Crohn's disease. Qantitative fecal lekocyte excretion flfills some of the criteria for the ideal measrement of disease activity in Crohn's disease. It is a direct assessment of acte bowel inflammation and is not affected by the common extraintestinal complications sch as arthritis or liver disease. Frthermore, most intercrrent illnesses will not elevate fecal gralll).locyte loss, althogh both gastrointestinal bleeding and spprative lng disease may increase bowel l11in loss. Becase the half-life of l11in is 67 h, measrements can be repeated within a week withot the problem of excessive backgrond activity. An important consideration in the se of this techniqe to qantitate granlocytic loss is that the labeled granlocytes have normal fnction. In vitro tests of granlocyte fnction showed no detrimental effect of 111 In labeling and are in agreement with previos stdies (19,21). The maintenance of granlocytes in plasma dring the separation and labeling procedre with 111 In-tropolonate may offer an advantage over ll1in-oxine or ll1in-acetylacetonate labeling as the potentially cell-damaging step of plasma deprivation (22) is avoided. Althogh we can demonstrate no difference on in vitro testing between plasma-labeled and bffer-labeled cells (Pin- % Fecal excretion I CDAI o o Pre Post Pre Post Figre 5. Serial fecal granlocyte excretion and Crohn's disease activity index (CDAI) in 8 patients who improved on treatment.

6 1338 SAVERYMUTTU ET AL. GASTROENTEROLOGY Vol. 85, No. 6 ching AJ, npblished observations), in preliminary stdies we have demonstrated that the initial plmonary seqestration associated with bffer-labeled cells (17,19) that may reflect cell damage (23) is abolished by plasma labeling (24) and is associated with early accmlation in sites of inflammation (25) within 1 min of reinjection of the cells (npblished observations). Frther evidence to spport normal granlocyte fnction is provided by the halflife estimation of 6.8 h, similar to the vale of 7.6 h obtained sing [3H]thymidine labeling (26). A possible improvement in the method of calclating granlocyte excretion wold be expressing lllin excretion as a percentage of the 3-min recovery vale with the aim of allowing for variations in circlating granlocytes de to variations in the damage to granlocytes sstained dring labeling and for individal differences in the marginating pool de to spleen size. A major problem with this approach is the wide range of recovery vales even in normal sbjects, both in magnitde [e.g. sing lllin from 22%-41% (19) or 51Cr from 12%-126% (27)] and in the time to maximm recovery. Ths, Weiblen et al. (19) fond that this time varied between 1 min and 2 h. Frthermore, becase there is almost immediate ptake in sites of inflammation with 111 In-tropolonate granlocytes, granlocyte recovery wold be nderestimated in patients with active disease. Finally, one cannot assme that noncirclating granlocytes are not available fot fecal excretion. Seqential gamma scanning between 4 min and 24 h after reinjection of 111 In-granlocytes has demonstrated a decrease of p to 7% of initial hepatic activity and p to 6% of initial splenic activity (npblished observations), probably representing a retrn to the circlation of both reversibly damaged granlocytes and marginated granlocytes, which presmably are available for fecal excretion. The validity of this techniqe in assessing fecal granlocyte loss in Crohn's disease necessitates the stable labeling of 111 In to granlocytes and no reabsorption of 111 In from cells or cell fragments in the bowel lmen. As one wold expect from the avid intracelllar binding of lllin (28), we cold demonstrate little evidence of eltion in vitro; this finding is in agreement with other workers who have stdied eltion for periods of p to 24 h (19,29). In vivo eltion appears nlikely becase plasma levels of 111 In (bond to transferrin) do not rise over the first 48 h. Similar findings have previosly been reported (19). Stool analysis sggests that at least 8% of ll1in activity is still in intact lekocytes or attached particlate fragments in feces. The remaining 2% may also be in a particlate fraction too small to sediment at 2 g. Alternatively, this may represent l11in_ transferrin that has leaked into the bowel. It is nlikely, however, that this cold accont for all of this activity, as the maximm circlating free lllin activity after 4 min from reinjection was 6%. Frther evidence spporting the stable in vivo binding of 111 In to lekocytes is provided by skin window stdies demonstrating the parallel accmlation of lekocytes and cell-bond radioactivity after reinjection of lllin-lekocytes (3). In animal stdies, the absorption of 111 In bond to transferrin after oral administration has been shown to be negligible (31). Becase there is negligible absorption of 111 In after oral administration of lllin-granlocytes in hmans, fecal l11in excretion shold not nderestimate ll1in_ granlocyte excretion. Althogh pre granlocyte separation and labeling involves additional expertise, the se of sch a preparation offers several advantages over the conventional mixed lekocyte preparation. A smaller radiation dose may be given, and the possible damaging effects on lymphocytes are avoided. Bowel images are clearer, and the range offecal excretion is greater. The higher fecal excretion with lllin-granlocytes than with mixed lekocytes is de to a nmber of factors. Any lymphocyte in the mixed cell preparation wold be damaged by the radiation dose sed (32) and so wold be navailable for migration. Althogh the inflammatory infiltrate in Crohn's disease in remission is primarily lymphocytic, with relapse the proportion of granlocyte markedly increases and the predominant cell type in the exdate is the granlocyte. Becase, in the mixed lekocyte preparation, only 31 % of the 111 In is on the granlocytic fraction, fecal indim excretion wold be expected to be less with the lekocyte preparation than with the granlocyte preparation. Qantitative fecal lekocyte excretion provides a novel approach to the assessment of disease activity in Crohn's disease and shold prove sefl in the detailed evalation of therapies and as a standard for the development of alternative methods of assessment. References 1. Best WR, Becktel JM, Singleton JW, Kern F. Development of the Crohn's disease activity index. Gastroenterology 1976; 7: Andre C, Descos L, Landais P, Fermanian J. Assessment of appropriate laboratory measrement to spplement the Crohn's disease activity index. Gt 1981;22: Cooke WT, Fowler DC, Cox EV, et al. The clinical significance of seromcoids in regional ileitis and lcerative colitis. Gastroenterology 1958;34: Fagan EA, Dyck RF, Maton PN, Hodgson HJF, Chadwick VS, Pepys MB. Serm levels of C-reactive protein in Crohn's disease and lcerative colitis. Er J Clin Invest 1982;12:351-6.

7 December 1983 FECAL "'IN-LEUKOCYTE IN CROHN'S DISEASE Kafman S, Chalmer B, Heilman R, Beeken W. A prospective stdy of the corse of Crohn's disease. Am J Dig Dis 1979; 24: Fergson T. Allan RN. Cooke WT. A stdy of the celllar infiltrate of the proximal jejnal mcosa in lcerative colitis and Crohn's disease. Gt 1975;16: Goodeman SR. Skinner JM, Trelove Sc. Abnormalities in the apparently normal bowel mcosa in Crohn's disease. Lancet 1976;ii: Saverymtt SH, Peters AM. Lavender JP, Hodgson HJF, Chadwick VS. l11indim labelled atologos lecocytes in inflammatory bowel disease. Gastroenterology 1981;8: Segal AW, Ensell j, Mnro JA, Sarner M. l11indim tagged lecocytes in the diagnosis of inflammatory bowel disease. Lancet 1981;ii: Lockhart-Mmmery HE, Morson B1. Crohn's disease (regional enteritis) of the large intestine and its distinction from lcerative colitis. Gt 196;1: Lockhart-Mmmery HE, Morson BL. Crohn's disease of the large intestine. Gt 1964;5: International Committee for Standardization in Haematology. Reference method for the erythrocyte sedimentation rate (ESR): test on hman blood. Br J Haematol 1973;24: Pepys MB, Dash AC, Ashley J. Isolation of C-reactive protein by affinity chromatography. Clin Exp ImmnoI1977;3: Rbini ME, Sheehy TW. Exdative enteropathy. A comparative stdy of Cr 51 and [131 PVP. J Lab Clin Med 1964;58: Danpre HJ, Osman SO, Brady F. The labelling of blood cells in plasma with 111In-tropolonate. Br J Radiol 1982;55: McDogall IR, Bamert JE. Lantieri R1. Evalation of 1l1In lekocyte whole body scanning. Am J Roentgenol 1979;133: Thakr ML, Lavender JP, Arnot RN, Silvester OJ, Segal AW. Indim-Ill labelled atologos lecocytes in man. J Ncl Med 1977;18: Reba RC, Salkeld J. In vitro stdies of malabsorption and other GI disorders. Semin Ncl Med 1982;12: Weiblen BJ, Forstrom L, McCllogh J. Stdies of the kinetics of indim 111 labelled granlocytes. J Lab Clin Med 1979; 94: Beeken WL. Clearance of circlating radiochromat~d albmin and erythrocytes by the gastrointestinal tract of normal sbjects. Gastroenterology 1967;52: Zakhireh B, Thakr ML, Malech HL, Cohen MS, Gottschalk A, Rot RK. Indim-Ill-labelled hman polymorphonclear lekocytes: viability, random migration, chemotaxis, bactericidal capacity and ltrastrctre. J Ncl Med 1979;2: Talstad l. Protection of blood cells by plasma proteins. Acta Med Scand 1971;19: Thakr ML, Coleman RE, Welch MJ. Indim-Ill-labelled lekocytes for the localisation of abscesses: preparation, analysis, tisse distribtion and comparison with gallim-67 citrate in dogs. J Lab Clin Med 1977;89: Peters AM, Saverymtt SH, Danpre H, Osman S, Reavy H. Lavender JP, Hodgson Hj, Chadwick VS. Granlocyte kinetics: isolation and labelling in plasma abolishes lng seqestration (abstr). Clin Sci 1982;62:7p. 25. Saverymtt SH, Peters AM, Reavy H, Lavender JP, Hodgson HJ, Chadwick VS. Avoidance of plmonary seqestration of l11in labelled lecocytes permits early diagnosis of inflammatory foci (abstr). Er J Clin Invest 1982;12: Dancey JT, Debelbeiss KA, Harker LA, Finch CA. Netrophil kinetics in man. J Clin Invest 1976;58: Dresch C, Najean Y, Bachet J. Kinetic stdies of 51Cr and Dp 2 P labelled granlocytes. Br J Haematol 1975;29: Thakr ML, Segal AW, Lois L, Welch MJ, Hopkins J, Peters TJ. Indim-Ill-labelled celllar blood components: mechanism of labelling and intracelllar location in hman netrophils. J Ncl Med 1977;18: Brke JET, Roath S, Ackery, Wyeth P. The comparison of 8 hydroxyqinoline, tropolone, acetylacetone as mediators in the labelling of polymorphonclear lecocytes with indim A fnctional stdy. Er J Ncl Med 1982;7: Wanda II JH. Lecocyte mobilisation to skin lesions. Acta Pathol Microbiol Scand 198;88: Toya JJ, Anselmi OE, Riley RE. Bennett LR. Metabolism of l11indim transferrin as a measre of bone marrow activity. In: Dynamic stdies with radioisotopes in medicine. Vol 1. Vienna: IAEA; 1975: Chisholm PM, Petflrs AM. The effect of indim-ill labelling on the recirclation of rat lymphocytes. In: Thakr ML. Gottdchalk A, eds. Indim-Ill labelled netrophils. platelets and lymphocytes. New York: Trivinm, 1982.