RENAL FUNCTION AND PHOSPHORUS EXCRETION AFTER HUMAN RENAL HOMOTRANSPLANTATION

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myeloma was responsible in 54. 23 of these were lymphomas of the lymphosarcoma or reticulum-cell sarcoma type. It seems possible that some, perhaps many, of the 23 were actually anaplastic, atypical myelomas. Clinical observations must ultimately lead to the laboratory in order to test hypotheses. We suggest that in atypical cases such as these immunoprotein studies should other than

be done as in our fourth case, in order to show whether or not an unusual lesion of anaplastic appearance contains a homogeneous immunoglobulin. If it does, it makes more sense to us to consider this as a manifestation of myeloma rather than a second entirely different process.

Summary with myeloma had clinical features generally considered most unusual for this disease. 3 had effusion in a body cavity, 2 had multiple skin nodules, 2 had massive hepatomegaly not due to amyloid, and 1 each had multiple pulmonary nodules and occlusion of a large artery. The clinician, pathologist, and immunochemist must be aware of the possibility of bizarre findings such as these in the natural history of myeloma if they are to make correct diagnoses and plan rational therapy. This work was supported in part by a grant from the American 4

patients

Cancer

Society.

Requests for reprints should be addressed to J. R. D., 3401, North Broad Street, Philadelphia, Pennsylvania, U.S.A. REFERENCES

Durant, J. R., Joseph, R. R., Tassoni, E., Zarafonetis, C. J. D. Unpublished Innes, J., Newall, J. (1961) Lancet, i, 239. Lieberman, P. H., Rosvoll, R. V., Ley, A. B. (1965) Cancer, 18, 727. Osserman, E. F., Takatsui, K. (1963) Medicine, 42, 357.

RENAL FUNCTION AND PHOSPHORUS EXCRETION AFTER HUMAN RENAL HOMOTRANSPLANTATION ROGER C. HERDMAN

ALFRED F. MICHAEL

M.D. Yale

M.D.

POSTDOCTORAL RESEARCH

FELLOW,

ESTABLISHED

Temple INVESTIGATOR,

U.S. PUBLIC HEALTH SERVICE

AMERICAN HEART ASSOCIATION

ROBERT L. VERNIER

WILLIAM D. KELLY M.D., Ph.D. Minnesota

M.D. Cincinnati ESTABLISHED

INVESTIGATOR,

AMERICAN HEART ASSOCIATION

CAREER DEVELOPMENT

AWARDEE,

U.S. PUBLIC HEALTH SERVICE

ROBERT A. GOOD M.D., Ph.D. Minnesota AMERICAN LEGION MEMORIAL HEART RESEARCH PROFESSOR OF PEDIATRICS AND MICROBIOLOGY

From the Pediatric Research Laboratories of the Variety Club Heart Hospital, the Department of Pediatrics, and the Department of Surgery, University of Minnesota

EARLY in

experience with human renal homothe University of Minnesota, we encountered a clinical problem which stimulated us to investigate the function of transplanted human kidneys intensively. The following is a summary of this case. our

transplantation

at

Case-report On Aug. 9, 1963, a 16-year-old white female was admitted to the University of Minnesota Hospitals with terminal chronic glomerulonephritis. On Sept. 28, 1963, she underwent bilateral nephrectomy and splenectomy. At the same time two cadaver kidneys were placed in the iliac fossx by standard techniques. After a stormy postoperative course, the patient was discharged on Dec. 7, 1963, to be kept under observation as an outpatient. Serum-calcium in hospital had ranged from 8-4 to 12-5 mg. per 100 ml. and serum-phosphorus had been 1-8-6-4 mg. per 100 ml. She was given ’Imuran’ (azathioprine) and ’Fura-

dantin ’ (nitrofurantoin). A week after discharge she was put low-calcium diet but because ofcontinuing hypercalcsemia and a drop in standard creatinine-clearance from 90 litres per day to 33 litres per day, she was readmitted on Dec. 22, 1963. On admission, tubular reabsorption of phosphorus (T.R.P.) was 72-0% at a time when serum-calcium was 11-7mg. per 100 ml. and phosphorus was 5-6 mg. per 100 ml. We thought she was undergoing a homograft rejection and cortisone therapy was started. While in hospital, her renal function improved and her hypercalcasmia abated. She was discharged and cortisone was gradually withdrawn. Serum-calcium and phosphorus were 9-8 and 2-3 mg. per 100 ml., respectively, and her diet was restricted only as to salt. On Feb. 17, 1964, however, she was readmitted with lethargy, vomiting, second-degree heart-block, and serum-calcium of 18-8 mg. per 100 ml. and phosphorus of 3-6 mg. per 100 ml. T.R.P. at that time was 75-6%. Because of failing renal function, which seemed to follow rather than precede her hypercalcasmia, the steroid dosage was again increased to 90 mg. prednisone a day. At this dose, hypercalcasmia and renal function improved. on a

On March 11, 1964, however, three parathyroid glands were removed during surgical exploration. One of these was normal but the other two, measuring 3 and 5 mm. in diameter, contained solid sheets of small chief cells compatible with secondary hyperparathyroidism. The patient was later discharged but has been kept under observation. She has continued on imuran and also prednisone 30 mg. every other day. Her creatinine clearances have remained in the range of 70-90 litres per day per 1-73 sq. m. and hypercalcxmia has not recurred.

Our tentative but unconfirmed diagnosis was that this patient with longstanding renal disease had secondary hyperparathyroidism which had become autonomous after renal function had been improved by kidney homografts. This case prompted us to examine glomerular filtrationrate (G.F.R.), renal blood-flow, and phosphorus excretion in other patients and we report here on renal function studies in 6 patients who had had kidney homografts after bilateral nephrectomy. Materials and Methods 6 patients who had had renal homotransplants in the University of Minnesota Hospitals were studied at varying intervals after transplantation while on the surgical and pasdiatric wards. The age and sex of these patients and the ischxmia time and time interval from transplantation to study are shown in table i. In each instance the patient’s own diseased kidneys were removed at the time of renal homotransplantation. Patients 1 and 2 had chronic pyelonephritis and patients 3-6 chronic glomerulonephritis. Kidneys were donated to patients 1-4 by the father (aged 42), mother (aged 41), sister (aged 29), and mother (aged 52) respectively. Patients 5 and 6 each received a kidney from female cadaver donors (aged 23 and 33 respectively) who had died while on the pump oxygenator during attempted surgical correction of cardiac defects. Thus all kidneys had been well perfused and oxygenated up to the moment of transfer. All patients received kidneys from ABO compatible donors. Patients 1-4 and 6 were receiving only intravenous fluids and patient 5 was on an unrestricted diet. All patients were receiving imuran; patients 5 and 6 were also receiving high doses of prednisone; patients 3-6 were receiving hydralazine 150, 100, 100, and 60 mg. per day respectively; patients 1 and 2 were not being treated with antihypertensive drugs. Patient 1 had a ureteral implantation to an ileal loop; the loop had a good flow without residual volume. Otherwise all patients had standard iliac-renal anastomoses and donorpelvis/host-ureter connection. No obstruction to urine flow was noted. Patients 1-4 had had no clinical evidence of rejection activity, patient 5 was in a chronic rejection phase, and patient 6 was recovering from an acute rejection with anuria. The donors for patients 1 and 4 were also investigated 48 hours after nephrectomy. Inulin and p-aminohippuric acid (P.A.H.) clearances were carried out according to standard techniques (Smith 1965)

122

using three consecutive 30-minute collection periods with blood-samples obtained at the midpoint of each urine collection. Except in patient 1 who had an ileostomy, urine was collected by urethral catheter at 1 or 2 days after transplantation and by spontaneous voiding 4, 10, and 19 days after transplantation. In an effort to turn off parathormone and decrease phosphorus excretion, standard calcium gluconate infusions (15 mg. calcium per kg.) were carried out in patients 2, 4, and 6 over a period of 4 hours, followed by four 4-hour phosphorus and creatinine clearances. All laboratory determinations were performed in duplicate: blood and urine were analysed for inulin and P.A.H. (Bojeson 1952, Smith 1956), creatinine (Peters and van Slyke 1932), phosphorus (Reiner 1953), and calcium (Appleton et al. 1959). Results

The results are tabulated in tablesI and 11. Inulin clearances in the patients following transplantation were lower than would be expected in a single kidney, especially when compared with the " control " values from the kidney remaining in the donor. The decrease in G.F.R. (as measured by inulin clearance) was proportionately much greater than the decrease in renal plasma-flow (measured by P.A.H. clearance); this is reflected by the strikingly low filtration fractions which vary between 0-060 and 0-149 in these 6 patients. Studies on the kidney remaining in each of two donors showed normal filtration fractions (table 11). Creatinine clearances were regularly higher than inulin clearances in the patient group as might be expected with serum-creatinines higher than normal. The level of serum-phosphorus was below normal in 2 patients, greater than normal in 2, and within normal limits in 2. The T.R.P. of all patients was extremely low (range 0-43-4%) in spite of a relatively good G.F.R. and renal plasma-flow (patient 3). Although there is some evidence that P.A.H. interferes with phosphorus reabsorption (Lewis and Ford 1961) we did not observe this during

Tubular reabsorption of phosphorus (’10) in patients 2, 4, and 6 before, during, and at four intervals after infusion of calcium

gluconate.

Phosphorus excretion was not inhibited suggesting that these patients, like the original one, had autonomous hyperparathyroidism. clearances where low levels of serum-P.A.H. were found (unpublished data, and see table 11). In 2 patients (5 and 6) more phosphorus was excreted than filtered in two of three collection periods. Calcium infusion studies in 3 patients (2, 4, and 6) resulting in increases of serum-calcium to 16-1, 13-1, and 15-6 mg. per 100 ml. respectively, did not produce any later increase in T.R.P. or fall in phosphorus clearance (see accompanying figure). Discussion of renal homograft function have not Early studies demonstrated the striking decreases in inulin clearance

TABLE I-RENAL FUNCTION AND PHOSPHORUS EXCRETION IN PATIENTS WHO HAVE UNDERGONE RENAL HOMOTRANSPLANTATION

Cin=Inulin clearance. Pe =Phosphorus excreted. Cpah=p-aminohippuric acid clearance. Cp=Phosphorus clearance. T.R.p.== Tubular reabsorption of phosphorus. F.F. = Filtration fraction. Ccr=Creatinine clearance. P{= Phosphorus altered. * Time of study indicates the lapse from time of transplantation and the ischeemia time refers to the interval between kidney removal from the donor to effective transplantation to the recipient. t T.R.P. and F.F. are calculated on the basis of inulin clearance. TABLE II-FUNCTION OF KIDNEY REMAINING IN DONOR PATIENTS

123

clearance described in this report al. (Tischler 1963, Ogden et al. 1965). Indeed, previous have shown little difference in function investigators between renal autotransplants and homotransplants and no characteristic functional changes in the human or animal homograft except a decline associated with rejection (Dossetor et al. 1963, Tischler et al. 1963, Murphy et al. 1964, Starzl et al. 1964, Ogden et al. 1965, Rosen et al. relative

to P.A.H. et

1965). Bricker et al. (1956) noted a slight decrease in the filtration fraction in an identical twin transplant. Striking alterations in inulin clearance relative to P.A.H. clearance as a result of anoxia or denervation have not been reported. Bricker and his colleagues felt that these factors might produce decreases in filtration fraction (Bricker et al. 1956, 1958) but other workers have not confirmed this (Nathan et al. 1962, Tischler et al. 1963, Miller et al. 1964). Other investigations have indicated that the rate of functional hyperplasia of a single kidney is quite slow (Burghele and Dimitriu 1963) and that if there is slight tubular predominance it is evident only by comparing G.F.R.P.A.H. tubular maximum ratios (Welsh et al. 1944, Michie et al. 1954, Kolberg 1959). Investigations of the denervated kidney have yielded somewhat contradictory results, perhaps because of failure of complete denervation in some studies; the changes in filtration-rate have not been striking (Forster and Maes 1947, Bricker et al. 1956, Miller et al. 1964), although moderate decreases in G.F.R. relative to renal plasma-flow have been reported (Bricker et al. 1958). In addition, striking alterations in circulatory distribution have not been noted in canine renal autografts (Rosen et al. 1965). The morphological picture of the transplanted kidney is one of near normal glomerular microscopy and early tubular and vascular abnormalities. These changes do not suggest an setiology for the relative decrease in G.F.R. If anatomical lesions of glomerular afferent arterioles arise shortly after kidney homografting-as suggested by the work of Darmady et al. (1964) and Porter et al. (1965)cortical glomerular circulation might decrease while bloodflow through juxtamedullary glomeruli and vasse rectae continued. Although true arteriovenous shunts probably do not exist to any extent in the kidney, such alterations in flow might provide an effective shunting of blood away from many glomeruli. It is difficult to see how this mechanism could explain normal clearances of P.A.H., however, since a shunt would also decrease blood-flow past proximal tubules where P.A.H. is extracted. Although the exact mechanism is unknown, clearances of P.A.H. varying from slightly decreased to increased, coincident with strikingly decreased G.F.R.S, could be explained by dilatation of glomerular efferent arterioles in the presence of normal or partially closed afferent arterioles. Lowering of filtration pressure and, thereby, filtration-rate without necessarily decreasing blood-flow is the net result of this alteration in hxmodynamics. The phosphorus data demonstrate consistently low tubular reabsorption of phosphorus, and, in patients 5 and 6, urinary excretion of more phosphorus than has been filtered. Though many workers have reported evidence for phosphorus secretion (Cooke et al. 1947, Barclay et al. 1949, Brodsky et al. 1956, Nicholson and Shepherd 1959, Scriver et al. 1964, Wilson et al. 1965), such a tubular function is not well established (Handler 1962, Strickler et al. 1964). In our patients, low rates of phosphorus reabsorption might be anticipated because of diminished

secondary hyperparathyroidism (Goldman and 1954) and high endogenous loads due to postoperative tissue breakdown and prolonged phosphorus retention in the pretransplantation period. Autonomous hyperparathyroidism is suggested in our patients by the failure of calcium infusion to inhibit phosphorus excretion (see accompanying figure). Furthermore, in the case reported here and also the one described by McPhaul et al. (1964), parathyroid surgery revealed the microscopic changes of parathyroid hyperplasia. Artificial depression of the filtration fraction and phosphorus loading have been used as a method of demonstrating phosphorus secretion (Barclay et al. 1949). According to Nicholson and Shepherd (1959), chemical lesions of the first part of the proximal tubule can inhibit reabsorption of phosphorus in the proximal tubule and uncover evidence of secretion in the distal tubule. Analogous degenerative changes in proximal tubules of human homografts have been demonstrated by microdissection (Darmady et al. 1964). We hope that this report will help physicians involved in the management of patients with renal homografts. Changes in filtration fraction which we believe are due to anomalies of renal perfusion may be susceptible to chemical manipulation. Early failure of perfusion could be ameliorated by paralysis of renal arterioles by the use of renal arterial infusions of papaverine or a similar agent. We have observed hypercalcsemia on several occasions in 3 other patients and it is evident that constant attention to parathyroid function is mandatory to avoid the damaging and dangerous effects of high levels of G.F.R.

and

Basset

serum-calcium.

Summary

Physiological studies of 6 human renal homografts have demonstrated a striking reduction in the filtration fraction stemming from a disproportionate decrease in glomerular filtration-rate. High phosphorus-clearances and very low tubular phosphorus reabsorption were apparent in patients studied 1-19 days after transplantation. In 2 patients phosphorus excretion exceeded filtered phosphorus during two of three clearance periods presenting evidence of actual tubular secretion of phosphorus. Calcium infusion had no effect on the high phosphorus clearance in 3 patients suggesting the presence of autonomous hyperparathyroidism. This work was aided by U.S. Public Health Service grants HE-05662, HE-06314, 2-F2-AM-19, AM-09007-01, AI-06063-02, and 212-03, by the American Heart Association, and by the National

Foundation.

Requests for reprints should be addressed to Prof. R. A. Good, Peediatric Research Laboratories of the Variety Club Heart Hospital, Minneapolis, Minnesota 55455, U.S.A. BIBLIOGRAPHY American Association of Clinical Chemists (1953) Standard Methods of Clinical Chemistry (edited by M. Reiner); vol. I, p. 84. New York. Appleton, H. D., West, M., Mandel, M., Salan, A. M. (1959) Clin. Chem.

5, 36. Barclay, J. A., Cooke, W. T., Kenney, R. A. (1949) Acta med. scand. 134, 107. Bojeson, E. (1952) ibid. suppl. 266, p. 275. Bricker, N. S., Guild, W. R., Reardan, J. B., Merrill, J. P. (1956) J. clin. Invest. 35, 1364. Straffon, R. A., Mahoney, E. P., Merrill, J. P. (1958) ibid. 37, 185. Brodsky, W. A., Shapiro, R. L., Kaim, J. T. (1956) Am. J. Physiol. 187, 589. Burghele, T., Dimitriu, D. (1963) Lyon Chir. 59, 641. Cooke, W. T., Barclay, J. A., Govan, A. D. T., Nagley, L. (1947) Archs intern. Med. 80, 147. Darmady, E. M., Offer, J. M., Stranack, F. (1964) Br. med. J. ii, 976. Dossetor, J. B., Morgen, R. O., Beck, J. C. (1963) Can. J. Biochem. Physiol. 41, 1409. Earle, D. P., Jr, Taggart, J. V., Shannon, J. A. (1944) J. clin. Invest. 23, 119. Forster, R. P., Maes, J. P. (1947) Am. J. Physiol. 150, 534. Goldman, R., Bassett, S. H. (1954) J. clin. Invest. 33, 1623. Handler, J. S. (1962) Am. J. Physiol. 202, 787. Harvey, R. B., Brothers, A. J. (1962) Ann. N. Y. Acad. Sci. 102, 46. Kolberg, A. (1959) Scand. J. clin. Lab. Invest. 11, suppl. 41, 11. Korner, P. I. (1963) Circulation Res. 12, 361. —

124

OUTPATIENT TREATMENT OF PSORIASIS A DOUBLE-BLIND TRIAL OF OINTMENTS C. F. ALLENBY SENIOR

M.A., M.B. Cantab., M.R.C.P. REGISTRAR, DEPARTMENT OF DERMATOLOGY, RADCLIFFE INFIRMARY, OXFORD

E. J. E. MUNRO-ASHMAN B.A., B.M. Oxon., M.R.C.P. R. S. WELLS M.D. Lond., M.R.C.P., M.R.C.P.E., D.C.H. FORMERLY

REGISTRARS, DEPARTMENT OF DERMATOLOGY, HOSPITAL, READING

ROYAL BERKSHIRE

OXFORD REGIONAL HOSPITAL

BOARD,

all be taken into consideration. Four treatments were used, of which two (A and C) were steroid preparations : must

A, 0-025% Fluocinolone acetonide ointment (’Synalar’, I.C.I.). B, the equivalent of a National Formulary prescription in a cosmetically acceptable base not previously used in this area (umz. oicis. salicvl.) :

A. BARR M.Sc. Belf., Ph.D. Reading STATISTICIAN,

quantitative assessment of the effect of the treatment required, for, with a relapsing condition such as psoriasis, the fact that a new preparation is comparable with older treatments does not automatically make it the treatment of choice. Cost, side-effects, and willingness and ability of the patient to undertake the treatment some

was

OXFORD

THE skin manifestations of psoriasis, affecting 1-2% of the population (Ingram 1954), appear in people with a genetic predisposition to the disease. Most patients are treated by their general practitioner (Calvert et al. 1963), but no permanent cure is yet available. The best results from outpatient treatment (MacLennan and Hellier 1961) are obtained when dithranol in Lassar’s paste is used after a tar bath and ultraviolet light, which gives clinical clearance of psoriasis in 95% of patients in an average of 19-5 days. Since this treatment is aathetically unpleasant, considerable effort is involved in persuading the patient to accept it. Like tar, mercury ointment stains, and Young (1960) found it no more effective than coal-tar ointment. The newer corticosteroid ointments are helpful, and their benefit is enhanced by occlusive, pliable, plastic films (Sulzberger and Witten 1961, Overton 1963, Williams et al. 1964), but they are expensive, and after long occlusion patients complain of smell, while folliculitis, miliaria (Shelley and Horvath 1950), reduced plasma-cortisol levels (Gill and Baxter 1964), and depression of anterior pituitary function (Kirketerp 1964), may occur. ’Alphosyl ’ (allantoin/coaltar cream) is cosmetically acceptable, but reports on its use (Bleiberg 1961, Welsh and Ede 1959, Clyman 1958) have not been based on double-blind clinical trials.

C, 0-025% Triamcinolone acetonide ointment (’Adcortyl E ’, Squibb). D, ’Alphosyl ’ cream (Stafford Miller).

Sample With the approval of their general practitioners, all patients with psoriasis who had attended the Radcliffe Infirmary, Oxford, or Royal Berkshire Hospital, Reading, in the previous five years were asked by letter whether they would like to take part in a trial of treatment if lesions were still present. One hundred and one accepted; most were not attending hospital and had had lesions for some years. Ten patients with psoriasis of guttate, arthropathic, exfoliating, or pustular-type were excluded. Nine others did not complete the trial, but as these were evenly distributed the results were unaffected. Eighty-two patients were available for assessment. Method On first attendance the lesions were recorded on a line diagram and a photograph of similar lesions on both sides of the body taken with a Leica camera IIIf using ’Kodachrome II’ TABLE I-SCORES AT CLINICAL ASSESSMENT

21 DAYS

AFTER TREATMENT

BEGAN

Design of Trial This trial was concerned primarily with different treatments which patients could undertake at home with little interference to their normal activities. At the same time

DR. HERDMAN AND OTHERS:

BIBLIOGRAPHY—continued

Lewis, J., Ford, R. V. (1961) J. Lab. clin. Med. 57, 547. McPhaul, J. J., Jr, McIntosh, D. A., Hammond, W. S., Park, O. K. (1964) New Engl. J. Med. 271, 1342. Michie, A. J., Michie, C. R., Ragni, M. C. (1954) Fedn Proc. Fedn Am. Socs exp. Biol. 13, 100. Miller, J., Pierce, J. C., Varco, R. L. (1964) Surgery, Gynec. Obstet. 119, 811. Murphy, G. P., Lawson, N. L., Johnston, G. S. (1964) Invest. Urol. 2, 261. Nathan, P., Foulkes, E. C., Wilchins, L. J., Miller, B. F. (1962) Proc. Soc. exp. Biol. Med. 111, 207. Nicholson, T. F., Shepherd, G. W. (1959) Can. J. Biochem. Physiol. 37, 103. Ogden, D. A., Sitprija, V., Holmes, J. H. (1965) Am. J. Med. 38, 873. Peters, S. P., van Slyke, D. D. (1932) Quantitative Clinical Chemistry

Methods; vol. II. Baltimore. Porter, K. A., Marchioro, T. C., Starzl, T. E. (1965) Br. J. Urol. 37, 250. Reubi, F. C. (1950) Proc. Soc. exp. Biol. Med. 73, 102. Rosen, S. M., Truniger, B., Kriek, H. R., Oken, D. E., Murray, J. E., Merrill, J. P. (1965) J. clin. Invest. 44, 1092. Scriver, C. R., Goldbloom, R. B., Roy, C. C. (1964) Pediatrics, Springfield, 34, 357. Smith, H. W. (1956) Principles of Renal Physiology. New York. Starzl, T. E., Marchioro, T. L., Porter, K. A., Moore, C. A., Rifkind, D., Waddell, W. R. (1964) Ann. intern. Med. 61, 470. Strickler, J. C., Thompson, D. D., Klose, R. M., Giebisch, G. (1964) J. Clin. Invest. 43, 1596. Tischler, V., Jacina, T., Gomboš, A., Skokan, I. (1963) Fedn Proc. Fedn Am. Socs exp. Biol. 22, T668, Welsh, C. A., Wellen, I., Taylor, H. C.. Jr. (1944) J. clin. Invest. 23, 750. Wilson, D. R., York, S. E., Jaworski, Z. F., Yendt, E. R. (1965) Medicine, Baltimore, 44, 99.

film and the ’Coronet P ’ electronic flash, fixed focus and fixed field of 15 x 10 cm. Treatment was dispensed by the pharmacists who gave each patient one ointment and indicated which side to occlude in accordance with a randomised block design. The patients were instructed to wash the lesions and apply the ointment twice a day, occluding one side with polyethylene secured with ’Sellotape’ for 12 hours overnight. The treatment given or the side occluded was not known to the observers. Each patient was seen after 21 days when the areas were re-photographed and changes assessed visually by a scoring system of 0 (no change); 1 (some improvement); 2 (much improved); 3 (clearance) under headings of redness, scaling, and size of lesions, giving a total possible score for complete clearance of 9. 4 weeks after the completion of the trial, pre- and post-treatment photographs of each patient were projected side by side on a large screen (no reference being made to the patient’s identity or previous assessment) and a new assessment recorded by each observer (CAi, RWJ. separately. This procedure was repeated 4 weeks later (CAi; RW2). A month later a joint assessment by both observers